Fibronectin based scaffold proteins having improved stability

ABSTRACT

The present application provides fibronectin based scaffold proteins associated with improved stability. The application also relates to stable formulations of fibronectin based scaffold proteins and the use thereof in diagnostic, research and therapeutic applications. The application further relates to cells comprising such proteins, polynucleotides encoding such proteins or fragments thereof, and to vectors comprising such polynucleotides.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.61/348,647, filed May 26, 2010, and 61/348,663, filed May 26, 2010,which applications are hereby incorporated by reference in theirentireties.

INTRODUCTION

Fibronectin based scaffolds are a family of proteins capable of evolvingto bind any compound of interest. These proteins, which generally makeuse of a scaffold derived from a fibronectin type III (Fn3) or Fn3-likedomain, function in a manner characteristic of natural or engineeredantibodies (that is, polyclonal, monoclonal, or single-chain antibodies)and, in addition, possess structural advantages. Specifically, thestructure of these antibody mimics has been designed for optimalfolding, stability, and solubility, even under conditions that normallylead to the loss of structure and function in antibodies. An example offibronectin-based scaffold proteins are Adnectins™ (Adnexus, a whollyowned subsidiary of Bristol-Myers Squibb).

Fibronectin is a large protein which plays essential roles in theformation of extracellular matrix and cell-cell interactions; itconsists of many repeats of three types (types I, II, and III) of smalldomains (Baron et al., 1991). Fn3 itself is the paradigm of a largesubfamily which includes portions of cell adhesion molecules, cellsurface hormone and cytokine receptors, chaperones, andcarbohydrate-binding domains. For reviews see Bork & Doolittle, ProcNatl Acad Sci USA. 1992 Oct. 1; 89(19):8990-4; Bork et al., J Mol Biol.1994 Sep. 30; 242(4):309-20; Campbell & Spitzfaden, Structure. 1994 May15; 2(5):333-7; Harpez & Chothia, J Mol Biol. 1994 May 13;238(4):528-39).

Fibronectin type III (Fn3) domains comprise, in order from N-terminus toC-terminus, a beta or beta-like strand. A; a loop, AB; a beta orbeta-like strand, B; a loop, BC; a beta or beta-like strand, C; a loop,CD; a beta or beta-like strand, D; a loop, DE; a beta or beta-likestrand, E; a loop, EF; a beta or beta-like strand, F; a loop, FG; and abeta or beta-like strand, G. Any or all of loops AB, BC, CD, DE, EF andFG may participate in target binding. The BC, DE, and FG loops are bothstructurally and functionally analogous to the complementaritydetermining regions (CDRs) from immunoglobulins. U.S. Pat. No. 7,115,396describes Fn3 domain proteins wherein alterations to the BC, DE, and FGloops result in high affinity TNFα binders. U.S. Publication No.2007/0148126 describes Fn3 domain proteins wherein alterations to theBC, DE, and FG loops result in high affinity VEGFR2 binders.

Protein pharmaceuticals may be associated with physical and chemicalinstability during their production, purification, storage and delivery.These instability issues can adversely impact the biological propertiesassociated with the protein therapeutic, thereby reducing the efficacyof that protein therapeutic. Sola, 2009, J. Pharm Sci, 98(4): 1223-1245.Accordingly, it would be advantageous to obtain improved fibronectindomain scaffold proteins that are associated with improved stability,e.g. reduced fragmentation and/or aggregation, that can be used for boththerapeutic and diagnostic purposes.

SUMMARY

One aspect of the application provides for novel fibronectin basedscaffold proteins that are associated with increased stability,including reduced fragmentation and/or reduced aggregation.

In some embodiments, the fibronectin based scaffold proteins providedherein comprise a fibronectin type III tenth (¹⁰Fn3) domain, wherein the¹⁰Fn3 domain comprises an amino acid sequence having at least 60%identity to SEQ ID NO: 1 and binds to a target molecule with a K_(D) ofless than 100 nM, and wherein the ¹⁰Fn3 domain further comprises aC-terminal tail that does not contain a DK sequence. In exemplaryembodiments, the C-terminal tail comprises the amino acid sequence ofSEQ ID NO: 4. In some embodiments, the C-terminal tail further comprisesa cysteine residue. In other embodiments, the C-terminal tail comprisesthe sequence of SEQ ID NO: 5.

In certain embodiments, the fibronectin based scaffold proteins bind toa target that is not bound by a wild-type ¹⁰Fn3 domain. In certainembodiments, fibronectin based scaffold proteins do not bind one or moreof EGFR, human serum albumin or PCSK9. In certain embodiments,fibronectin based scaffold proteins that comprise a single ¹⁰Fn3 domaindo not bind one or more of EGFR, human serum albumin or PCSK9. Incertain embodiments, multivalent fibronectin based scaffold proteins donot bind to one or more of the following combinations of targetmolecules: i) EGFR and IGF-IR; ii) EGFR and any other target protein; oriii) human serum albumin and any other target protein.

In some embodiments, the ¹⁰Fn3 domains of the fibronectin based scaffoldprotein further comprises an N-terminal extension comprising from 1-10amino acids. In other embodiments, the ¹⁰Fn3 domain of the fibronectinbased scaffold protein comprises a sequence selected from the groupconsisting of: M, MG, G, and any of SEQ ID NOs: 19-21 and 26-31.

In some embodiments, the fibronectin based scaffold proteins furthercomprise a second ¹⁰Fn3 domain, wherein the second ¹⁰Fn3 domaincomprises an amino acid sequence having at least 60% identity to SEQ IDNO: 1 and binds to a target molecule with a K_(D) of less than 100 nM,and wherein the second ¹⁰Fn3 domain further comprises a C-terminal tailthat does not contain a DK sequence. In exemplary embodiments, thesecond ¹⁰Fn3 domain comprises a C-terminal tail comprising the aminoacid sequence of SEQ ID NO: 4. In some embodiments, the first and second¹⁰Fn3 domains bind to different targets. In some embodiments, the second¹⁰Fn3 domain further comprises an N-terminal extension comprising from1-10 amino acids. In some embodiments, the N-terminal extensioncomprises a sequence selected from the group consisting of: M, MG, G,and any of SEQ ID NOs: 19-21 and 25-31. In some embodiments, the firstand second ¹⁰Fn3 domains are connected by a polypeptide linkercomprising from 1-30 amino acids. In some embodiments, the polypeptidelinker is selected from the group consisting of: a glycine-serine basedlinker, a glycine-proline based linker, a proline-alanine linker and aFn-based linker.

In certain embodiments, the fibronectin based scaffold protein comprisesone or more ¹⁰Fn3 domains comprising a loop, AB; a loop, BC; a loop, CD;a loop, DE; a loop, EF; and a loop, FG and each, independently, have atleast one loop selected from loop BC, DE, and FG with an altered aminoacid sequence relative to the sequence of the corresponding loop of thehuman ¹⁰Fn3 domain. In certain embodiments, the ¹⁰Fn3 domains comprisean amino acid sequence that is at least 50, 60, 70, or 80% identical tothe naturally occurring human ¹⁰Fn3 domain represented by SEQ ID NO: 1.In an exemplary embodiment, the fibronectin based scaffold protein is adimer comprising two ¹⁰Fn3 domains.

In another aspect, the application provides for novel fibronectin basedscaffold protein dimers that are associated with reduced proteinfragmentation as compared to the fibronectin based scaffold proteindimers described in PCT application WO 2009/142773. In some embodiments,the fibronectin based scaffold protein dimer comprises the amino acidsequence of SEQ ID NO: 48.

In another aspect, the application provides fibronectin based scaffoldprotein dimers having the structure N1-D1-C1-L-N2-D2-C2. In certainembodiment, N1 and N2 are optional N-terminal extensions independentlycomprising from 0-10 amino acids; D1 and D2 are independently selectedfrom the group consisting of: (I) a tenth fibronectin type 111 domain(¹⁰Fn3) domain having at least 95% identity with the amino acid sequenceset forth in SEQ ID NO: 2, wherein said ¹⁰Fn3 domain binds to IGF-IRwith a K_(D) of less than 500 nM, and (ii) a ¹⁰Fn3 domain having atleast 95% identity with the amino acid sequence set forth in SEQ ID NO:3, wherein said ¹⁰Fn3 domain binds to VEGFR2 with a K_(D) of less than500 nM; L is a polypeptide linker comprising from 0-30 amino acidresidues; C1 comprises the amino acid sequence set forth in SEQ ID NO:4; and C2 comprises the amino acid sequence set forth in SEQ ID NO: 4, 5or 6.

In some embodiments the fibronectin based scaffold protein dimers havethe structure N1-D1-C1-L-N2-D2-C2, wherein D1 comprises a ¹⁰Fn3 domainhaving at least 95% identity with the amino acid sequence set forth inSEQ ID NO: 2, and D2 comprises a ¹⁰Fn3 domain having at least 95%identity with the amino acid sequence set forth in SEQ ID NO: 3. Inother embodiments the fibronectin based scaffold protein dimers have thestructure N1-D1-C1-L-N2-D2-C2, wherein D1 comprises a ¹⁰Fn3 domainhaving at least 95% identity with the amino acid sequence set forth inSEQ ID NO: 3, and D2 comprises a ¹⁰Fn3 domain having at least 95%identity with the amino acid sequence set forth in SEQ ID NO: 2.

In some embodiments, the fibronectin based scaffold protein dimers havethe structure N1-D1-C1-L-N2-D2-C2, wherein C2 comprises the amino acidsequence of SEQ ID NO: 6.

In some embodiments, the fibronectin based scaffold protein dimers havethe structure N1-D1-C1-L-N2-D2-C2, wherein N1 comprises an amino acidsequence selected from the group consisting of: M, MG, G, and any one ofSEQ ID NOs: 19-21 and 26-31. In exemplary embodiments, N1 comprises theamino acid sequence of SEQ ID NO: 19. In some embodiments, thefibronectin based scaffold protein dimers have the structureN1-D1-CT-L-N2-D2-C2, wherein N2 comprises an amino acid sequenceselected from the group consisting of: M, MG, G, and any one of SEQ IDNOs: 19-21 and 26-31. In exemplary embodiments, N2 comprises the aminoacid sequence of SEQ ID NO: 20.

In some embodiments, the fibronectin based scaffold protein dimers havethe structure N1-D1-C1-L-N2-D2-C2, wherein L is a polypeptide linkerselected from the group consisting of: a glycine-serine based linker, aglycine-proline based linker, a proline-alanine linker and a Fn-basedlinker. In other embodiments, L comprises the amino acid sequence of SEQID NO: 7.

In some embodiments, the fibronectin based scaffold proteins furthercomprise one or more pharmacokinetic (PK) moieties selected from: apolyoxyalkylene moiety, a human serum albumin binding protein, sialicacid, human serum albumin, transferrin, IgG, an IgG binding protein, andan Fc fragment. In some embodiments, the PK moiety is a polyoxyalkylenemoiety and said polyoxyalkylene moiety is polyethylene glycol (PEG). Insome embodiments, the PEG moiety is covalently linked to the fibronectinbased scaffold protein via a Cys or Lys amino acid. In some embodiments,the PEG is between about 0.5 kDa and about 100 kDa.

In one aspect, the application provides pharmaceutically acceptablecompositions comprising a fibronectin based scaffold protein. In someembodiments, the composition is essentially pyrogen free. In someembodiments, the composition is substantially free of microbialcontamination making it suitable for in vivo administration. Thecomposition may be formulated, for example, for IV, IP or subcutaneousadministration. In some embodiments, the composition comprises aphysiologically acceptable carrier. In some embodiments, the pH of thecomposition is between 4.0-6.5. In some embodiments, the pH of thecomposition is between 4.0-5.5. In other embodiments, the pH of thecomposition is 5.5. In other embodiments, the pH of the composition is4.0. In some embodiments, the concentration of the fibronectin basedscaffold protein is 5 mg/ml in the composition.

In another aspect, the application provides a pharmaceutical formulationcomprising a fibronectin based scaffold protein, wherein the formulationcomprises at least 5 mg/ml of the fibronectin based scaffold protein,has a pH of 4.0, and is suitable for intravenous administration. In someembodiments, the pharmaceutical formulation is stable for at least 4weeks at 25° C. In some embodiments, the pharmaceutical formulation hasless than 4% fragmentation. In some embodiments, the formulation hasless than 4% aggregates.

In another aspect, the application provides a method for treating ahyperproliferative disorder in a subject comprising administering to asubject in need thereof a therapeutically effective amount of any of thecompositions as described herein.

In another aspect, the application provides a nucleic acid encoding afibronectin based scaffold protein as described herein. Vectorscontaining polynucleotides for such proteins are included as well.Suitable vectors include, for example, expression vectors. A furtheraspect of the application provides for a cell, comprising apolynucleotide, vector, or expression vector, encoding a fibronectinbased scaffold protein. Sequences are preferably optimized to maximizeexpression in the cell type used. In some embodiments, expression is ina bacterial cell. In other embodiments, expression is in a mammaliancell. Preferably, expression is in E. coli. In one aspect, the cellexpresses a fibronectin based scaffold protein. In one aspect, thepolynucleotides encoding fibronectin based scaffold proteins are codonoptimized for expression in the selected cell type. Also provided aremethods for producing a fibronectin based scaffold protein as describedherein, comprising culturing a host cell comprising a nucleic, vector,or expression vector, comprising a nucleic acid encoding the fibronectinbased scaffold protein and recovering the expressed fibronectin basedscaffold protein from the culture.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Size exclusion-high pressure liquid chromatography (SE-HPLC)data showing the amount of protein aggregation over time for twoconcentrations of pegylated V/I(DK+) (SEQ ID NO: 22). The pegylatedprotein was stored at 4° C. for 12 months in 10 mM succinic acid, 5%sorbitol, pH 5.5 at 3 mg/mL protein concentration.

FIG. 2. SE-HPLC data showing the amount of protein fragmentation overtime for two concentrations of pegylated V/I(DK+) (SEQ ID NO: 22). Thepegylated protein was stored at 4° C. for 12 months in 10 mM succinicacid, 5% sorbitol, pH 5.5 at 3 mg/mL protein concentration.

FIG. 3. SE-HPLC data showing the effect of pH on protein aggregationover time for pegylated V/I(DK+) (SEQ ID NO: 22). The pegylated proteinwas stored at 25° C. for 3 weeks in a formulation containing 50 mM NaCl,with 20 mM sodium acetate (for pH 4 and 5) or 20 mM sodium phosphate(for pH 6 and 7).

FIG. 4. SE-HPLC data showing the effect of pH on protein fragmentationover time for pegylated V/I(DK+) (SEQ ID NO: 22). The pegylated proteinwas stored at 25° C. for 3 weeks in a formulation containing 50 mM NaCl,with 20 mM sodium acetate (for pH 4 and 5) or 20 mM sodium phosphate(for pH 6 and 7).

FIG. 5. Reverse phase-high pressure liquid chromatography (RP-HPLC)profile showing the sites of cleavage of pegylated V/I(DK+) molecule(SEQ ID NO: 22) after storage at 25° C. for 4 weeks in 10 mM acetate,150 mM NaCl at pH 5.5. As indicated in the figure, protein cleavageoccurs directly following positions D95, D106, D180 and D200, withcleavage predominantly occurring directly following sites D95 and D200.The pegylated protein was stored for 4 week at 25° C. in 10 mM sodiumacetate, 150 mM sodium chloride, pH 5.5 at 5 mg/mL proteinconcentration.

FIG. 6. SE-HPLC data showing the effect of pH on protein aggregationover time for the pegylated E/I(DK+) (SEQ ID NO: 23). The pegylatedprotein was stored for 4 week at 25° C. in 10 mM succinic acid, 5%sorbitol, pH 4.0, 4.5 and 5.5.

FIG. 7. SE-HPLC data showing the effect of pH on the amount of proteinfragmentation over time for the pegylated E/I(DK t) (SEQ ID NO: 23). Thepegylated protein was stored for 4 week at 25° C. in 10 mM succinicacid, 5% sorbitol, pH 4.0, 4.5 and 5.5.

FIG. 8. RP-HPLC profile showing the sites of cleavage of the pegylatedE/I(DK+) (SEQ ID NO: 23) after storage at 25° C. for 4 weeks in 10 mMsuccinic acid, 5% sorbitol, pH 4.0, 5 mg/mL protein concentration. Asindicated in the figure, protein cleavage occurs directly followingpositions D95, D199 and D218.

FIG. 9. SE-HPLC data demonstrating showing the effect of pH on proteinaggregation over time for various fibronectin based scaffold proteinconstructs. The level of aggregation for various fibronectin basedscaffold proteins was tested at either pH 5.5 or pH 4.0 during storagefor 4 weeks at 25° C. E/I(DK+) is SEQ ID NO: 23; E/I(DK−, no C-term) isSEQ ID NO: 24; E/I(2DK−) is SEQ ID NO: 25; and V/I(DK+) is SEQ ID NO:22.

FIG. 10. RP-HPLC data showing the amount of fragmentation of variousfibronectin based scaffold proteins at different pHs during storage for4 weeks at 25° C. Fibronectin based scaffold proteins that contain DKsequences are more susceptible to fragmentation at pH 4.0 as compared topH 5.5. Fibronectin based scaffold proteins that do not contain DKsequences are more resistant to fragmentation at pH 4.0 as compared tofibronectin based scaffold proteins that contain DK sequences. E/I(DK+)is SEQ ID NO: 23; E/I(DK−, no C-term) is SEQ ID NO: 24; E/I(2DK−) is SEQID NO: 25; and V/I(DK+) is SEQ ID NO: 22.

FIG. 11. LC-MS data demonstrating that the major cleavage site in theE/I(DK−, no C-term) molecule (SEQ ID NO: 24) is D199, while the majorcleavage site in the E/I(DK+) molecule (SEQ ID NO: 23) is D218.

FIG. 12. LC-MS data demonstrating that the major cleavage site in theE/I(2DK−) molecule (SEQ ID NO: 25) is D199.

FIG. 13. The rates of aggregation observed in VI(DK+) (SEQ ID NO: 56)and VI(DK−) (SEQ ID NO: 57), under 25° C. storage for up to two months,as assessed by SE-HPLC analysis.

FIG. 14. Clip rates observed in VI(DK+) (SEQ ID NO: 56) and VI(DK−) (SEQID NO: 57), under 25° C. storage for up to two months, as assessed byRP-HPLC analysis.

FIG. 15. Clip region of a RP-HPLC overlaid chromatogram of VI(DK+) (SEQID NO: 56) and VI(DK−) (SEQ ID NO: 57), after incubation for 2 months at25° C. The total % clips for VI(DK+) is 16%, whereas for VI(DK−) it is6.9%.

DETAILED DESCRIPTION Definitions

By a “polypeptide” is meant any sequence of two or more amino acids,regardless of length, post-translation modification, or function.“Polypeptide,” “peptide,” and “protein” are used interchangeably herein.Polypeptides can include natural amino acids and non-natural amino acidssuch as those described in U.S. Pat. No. 6,559,126, incorporated hereinby reference. Polypeptides can also be modified in any of a variety ofstandard chemical ways (e.g., an amino acid can be modified with aprotecting group; the carboxy-terminal amino acid can be made into aterminal amide group; the amino-terminal residue can be modified withgroups to, e.g., enhance lipophilicity; or the polypeptide can bechemically glycosylated or otherwise modified to increase stability orin vivo half-life). Polypeptide modifications can include the attachmentof another structure such as a cyclic compound or other molecule to thepolypeptide and can also include polypeptides that contain one or moreamino acids in an altered configuration (i.e., R or S; or, L or D).

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are obtained as describedbelow by using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc. has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087, and is publicly available throughGenentech, Inc., South San Francisco, Calif. The ALIGN-2 program shouldbe compiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows: 100times the fraction X/Y where X is the number of amino acid residuesscored as identical matches by the sequence alignment program ALIGN-2 inthat program's alignment of A and B, and where Y is the total number ofamino acid residues in B. It will be appreciated that where the lengthof amino acid sequence A is not equal to the length of amino acidsequence B, the % amino acid sequence identity of A to B will not equalthe % amino acid sequence identity of B to A.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thedisorder. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy in vivo can, for example, be measured by assessing the time todisease progression (TTP) and/or determining the response rates (RR).

The half-life of an amino acid sequence or compound can generally bedefined as the time taken for the serum concentration of the polypeptideto be reduced by 50%, in vivo, for example due to degradation of thesequence or compound and/or clearance or sequestration of the sequenceor compound by natural mechanisms. The half-life can be determined inany manner known per se, such as by pharmacokinetic analysis. Suitabletechniques will be clear to the person skilled in the art, and may forexample generally involve the steps of suitably administering to theprimate a suitable dose of the amino acid sequence or compound to betreated; collecting blood samples or other samples from said primate atregular intervals; determining the level or concentration of the aminoacid sequence or compound of the invention in said blood sample; andcalculating, from (a plot of) the data thus obtained, the time until thelevel or concentration of the amino acid sequence or compound of theinvention has been reduced by 50% compared to the initial level upondosing. Reference is for example made to the standard handbooks, such asKenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook forPharmacists and in Peters et al, Pharmacokinete analysis: A PracticalApproach (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi& D Perron, published by Marcel Dekker, 2nd Rev. edition (1982).

Half-life can be expressed using parameters such as the t1/2-alpha,t½-beta and the area under the curve (AUC). In the presentspecification, an “increase in half-life” refers to an increase in anyone of these parameters, such as any two of these parameters, oressentially all three these parameters. An “increase in half-life” inparticular refers to an increase in the t1/2-beta, either with orwithout an increase in the t½-alpha and/or the AUC or both.

Overview

The present application describes improved fibronectin based scaffoldproteins that are associated with increased stability. The fibronectinbased scaffold proteins described herein comprise one or more humantenth fibronectin type III domains that have been modified so as to bindto one or more desired targets. The present application also describesimproved VEGFR2/IGF-IR bispecific fibronectin based scaffold proteindimers that are associated with increased stability, comprising twohuman tenth fibronectin type III domains, one that has been modified soas to bind specifically to VEGFR2 and one that has been modified so asto bind specifically to IGF-IR. PCT application WO 2009/142773 describesfibronectin scaffold multimers that may be linked covalently ornon-covalently and that bind both VEGFR2 and IGF-IR. The presentapplication relates, in part, to the surprising discovery thatbispecific fibronectin based scaffold proteins that bind to VEGFR2 andIGF-IR experience a high frequency of fragmentation at certain aspartateresidues. In particular, it has been discovered that aspartate residuesdirectly followed by a lysine residue are more sensitive to cleavage infibronectin based scaffold proteins as compared to aspartate residuesfollowed by other amino acids. The application also relates to thesurprising discovery that the degree of fragmentation of VEGFR2/IGF-IRbinding fibronectin based scaffold proteins is considerably higher thanthe degree of fragmentation associated with a related fibronectin basedscaffold protein, i.e., a bispecific EGFR/IGF-IR binding fibronectinbased scaffold protein dimer, despite identical storage conditions and ahigh percentage of shared sequence identity between these similarproteins. The application also demonstrates that fragmentation of amodel fibronectin based scaffold protein can be markedly reduced if theDK sites are removed or modified to substitute the aspartate residue fora different amino acid, e.g. glutamic acid. Also provided herein areimproved compositions of fibronectin based scaffold proteins that haveincreased stability during storage.

Fibronectin Based Scaffolds

Fn3 refers to a type III domain from fibronectin. An Fn3 domain issmall, monomeric, soluble, and stable. It lacks disulfide bonds and,therefore, is stable under reducing conditions. The overall structure ofFn3 resembles the immunoglobulin fold. Fn3 domains comprise, in orderfrom N-terminus to C-terminus, a beta or beta-like strand, A; a loop,AB; a beta or beta-like strand, B; a loop, BC; a beta or beta-likestrand, C; a loop, CD; a beta or beta-like strand, D; a loop, DE; a betaor beta-like strand, E; a loop, EF; a beta or beta-like strand, F; aloop, FG; and a beta or beta-like strand, G. The seven antiparallelβ-strands are arranged as two beta sheets that form a stable core, whilecreating two “faces” composed of the loops that connect the beta orbeta-like strands. Loops AB, CD, and EF are located at one face andloops BC, DE, and FG are located on the opposing face. Any or all ofloops AB, BC, CD, DE, EF and FG may participate in ligand binding. Thereare at least 15 different modules of Fn3, and while the sequencehomology between the modules is low, they all share a high similarity intertiary structure.

Adnectins™ (Adnexus, a Bristol-Myers Squibb Company) are ligand bindingscaffold proteins based on the tenth fibronectin type III domain, i.e.,the tenth module of Fn3, (¹⁰Fn3). The amino acid sequence of thenaturally occurring human ¹⁰Fn3 is set forth in SEQ ID NO: 37:VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 37) (the AB, CD and EFloops are underlined, and the BC, FG, and DE loops are emphasized inbold).

In SEQ ID NO: 37, the AB loop corresponds to residues 15-16, the BC loopcorresponds to residues 21-30, the CD loop corresponds to residues39-45, the DE loop corresponds to residues 51-56, the EF loopcorresponds to residues 60-66, and the FG loop corresponds to residues76-87. See e.g., Xu et al., Chemistry & Biology 2002 9:933-942. The BC,DE and FG loops align along one face of the molecule and the AB, CD andEF loops align along the opposite face of the molecule. In SEQ ID NO:37, beta strand A corresponds to residues 9-14, beta strand Bcorresponds to residues 17-20, beta strand C corresponds to residues31-38, beta strand D corresponds to residues 46-50, beta strand Ecorresponds to residues 57-59, beta strand F corresponds to residues67-75, and beta strand G corresponds to residues 88-94. The strands areconnected to each other through the corresponding loop, e.g., strands Aand B are connected via loop AB in the order: strand A, loop AB, strandB, etc. The first 8 amino acids of SEQ ID NO: 37 (italicized above) maybe deleted while still retaining binding activity of the molecule.Residues involved in forming the hydrophobic core (the “core amino acidresidues”) in SEQ ID NO: 37 include the amino acids corresponding to thefollowing amino acids of SEQ ID NO: 37: L8, V10, A13, L18, I20, W22,Y32, I34, Y36, F48, V50, A57, I59, L62, Y68, I70, V72, A74, I88, I90 andY92, wherein the core amino acid residues are represented by the singleletter amino acid code followed by the position at which they arelocated within SEQ ID NO: 37. See e.g., Dickinson et al., J. Mol. Biol.236: 1079-1092 (1994).

¹⁰Fn3 domains are structurally and functionally analogous to antibodies,specifically the variable region of an antibody. While ¹⁰Fn3 domains maybe described as “antibody mimics” or “antibody-like proteins”, they dooffer a number of advantages over conventional antibodies. Inparticular, they exhibit better folding and thermostability propertiesas compared to antibodies, and they lack disulphide bonds, which areknown to impede or prevent proper folding under certain conditions.

The BC, DE, and FG loops of ¹⁰Fn3 domains are analogous to thecomplementary determining regions (CDRs) from immunoglobulins.Alteration of the amino acid sequence in these loop regions changes thebinding specificity of ¹⁰Fn3. ¹⁰Fn3 domains with modifications in theAB, CD and EF loops may also be made in order to produce a molecule thatbinds to a desired target. The protein sequences outside of the loopsare analogous to the framework regions from immunoglobulins and play arole in the structural conformation of the ¹⁰Fn3. Alterations in theframework-like regions of ¹⁰Fn3 are permissible to the extent that thestructural conformation is not so altered as to disrupt ligand binding.Methods for generating ¹⁰Fn3 ligand specific binders have been describedin PCT Publication Nos. WO 00/034787, WO 01/64942, and WO 02/032925,disclosing high affinity TNFα binders, PCT Publication No. WO2008/097497, disclosing high affinity VEGFR2 binders, PCT PublicationNo. WO 2008/066752, disclosing high affinity IGF-IR binders, and WO2009/142773, disclosing high affinity multivalent VEGFR2/IGF-IR binders.Additional references discussing ¹⁰Fn3 binders and methods of selectingbinders include PCT Publication Nos. WO 98/056915, WO 02/081497, and WO2008/031098 and U.S. Publication No. 2003/0186385.

As described above, amino acid residues corresponding to residues 21-30,51-56, and 76-87 of SEQ ID NO: 37 define the BC, DE and FG loops,respectively. However, it should be understood that not every residuewithin the loop region needs to be modified in order to achieve a ¹⁰Fn3binding domain having strong affinity for a desired target, such asVEGFR2 or IGF-TR. For example, in some embodiments, only residuescorresponding to amino acids 23-29 of the BC loop, 52-55 of the DE loop,and 77-86 of the FG loop were modified to produce high affinity ¹⁰Fn3binders (see e.g., the VEGFR2 binding core having SEQ ID NO: 3).Accordingly, in certain embodiments, the BC loop may be defined by aminoacids corresponding to residues 23-29 of SEQ ID NO: 37, the DE loop maybe defined by amino acids corresponding to residues 52-55 of SEQ ID NO:37, and the FG loop may be defined by amino acids corresponding toresidues 77-86 of SEQ ID NO: 37.

Additionally, insertions and deletions in the loop regions may also bemade while still producing high affinity ¹⁰Fn3 binding domains. Forexample, the FG loop of the VEGFR2 binder having SEQ ID NO: 3 has thesame length FG loop as the wild-type ¹⁰Fn3 domain, i.e., the 10 residues77-86 of SEQ ID NO: 37 were replaced with the ten residues 69-78 of SEQID NO: 3. In contrast, the FG loop of the IGF-IR binder having SEQ IDNO: 2 is shorter in length than the corresponding FG loop of thewild-type ¹⁰Fn3 domain, i.e., the 10 residues 77-86 of SEQ ID NO: 37were replaced with the six residues 69-74 of SEQ ID NO: 2. Finally, theFG loop of the EGFR binder having SEQ ID NO: 39 is longer in length thanthe corresponding FG loop of the wild-type ¹⁰Fn3 domain, i.e., the 10residues 77-86 of SEQ ID NO: 37 were replaced with the fifteen residues69-83 of SEQ ID NO: 39.

Accordingly, in some embodiments, one or more loops selected from BC,DE, and FG may be extended or shortened in length relative to thecorresponding loop in wild-type human ¹⁰Fn3. In some embodiments, thelength of the loop may be extended by from 2-25 amino acids. In someembodiments, the length of the loop may be decreased by 1-11 aminoacids. In particular, the FG loop of ¹⁰Fn3 is 12 residues long, whereasthe corresponding loop in antibody heavy chains ranges from 4-28residues. To optimize antigen binding, therefore, the length of the FGloop of ¹⁰Fn3 may be altered in length as well as in sequence to coverthe CDR3 range of 4-28 residues to obtain the greatest possibleflexibility and affinity in antigen binding. In some embodiments, one ormore residues of the integrin-binding motif “arginine-glycine-asparticacid” (RGD) (amino acids 78-80 of SEQ ID NO: 37) may be substituted soas to disrupt integrin binding. In one embodiment, the RGD sequence isreplaced by a polar amino acid-neutral amino acid-acidic amino acidsequence (in the N-terminal to C-terminal direction). In anotherembodiment, the RGD sequence is replaced with SGE.

The non-ligand binding sequences of ¹⁰Fn3, i.e., the “¹⁰Fn3 scaffold”,may be altered provided that the ¹⁰Fn3 domain retains ligand bindingfunction and/or structural stability. In some embodiments, one or moreof Asp 7, Glu 9, and Asp 23 are replaced by another amino acid, such as,for example, a non-negatively charged amino acid residue (e.g., Asn,Lys, etc.). These mutations have been reported to have the effect ofpromoting greater stability of the mutant ¹⁰Fn3 at neutral pH ascompared to the wild-type form (See, PCT Publication No. WO 02/04523). Avariety of additional alterations in the ¹⁰Fn3 scaffold that are eitherbeneficial or neutral have been disclosed. See, for example, Batori etal., Protein Eng. 2002 15(12): 1015-20; Koide et al., Biochemistry 200140(34): 10326-33.

The ¹⁰Fn3 scaffold may be modified by one or more conservativesubstitutions. As many as 5%, 10%, 20% or even 30% or more of the aminoacids in the ¹⁰Fn3 scaffold may be altered by a conservativesubstitution without substantially altering the affinity of the ¹⁰Fn3for a ligand. In certain embodiments, the scaffold may comprise anywherefrom 0-15, 0-10, 0-8, 0-6, 0-5, 0-4, 0-3, 1-15, 1-10, 1-8, 1-6, 1-5,1-4, 1-3, 2-15, 2-10, 2-8, 2-6, 2-5, 2-4, 5-15, or 5-10 conservativeamino acid substitutions. In exemplary embodiments, the scaffoldmodification preferably reduces the binding affinity of the ¹⁰Fn3 binderfor a ligand by less than 100-fold, 50-fold, 25-fold, 10-fold, 5-fold,or 2-fold. It may be that such changes will alter the immunogenicity ofthe ¹⁰Fn3 in vivo, and where the immunogenicity is decreased, suchchanges will be desirable. As used herein, “conservative substitutions”are residues that are physically or functionally similar to thecorresponding reference residues. That is, a conservative substitutionand its reference residue have similar size, shape, electric charge,chemical properties including the ability to form covalent or hydrogenbonds, or the like. Preferred conservative substitutions are thosefulfilling the criteria defined for an accepted point mutation inDayhoff et al., Atlas of Protein Sequence and Structure 5:345-352 (1978& Supp.). Examples of conservative substitutions are substitutionswithin the following groups: (a) valine, glycine; (b) glycine, alanine;(c) valine, isoleucine, leucine; (d) aspartic acid, glutamic acid; (e)asparagine, glutamine; (f) serine, threonine; (g) lysine, arginine,methionine; and (h) phenylalanine, tyrosine.

In one aspect, the application provides fibronectin based scaffoldproteins, e.g., polypeptides comprising at least one ¹⁰Fn3 domain andhaving a C-terminal tail that lacks a DK sequence. In exemplaryembodiments, the fibronectin based scaffold proteins comprise a ¹⁰Fn3domain having a C-terminal tail comprising the amino acid sequence ofSEQ ID NO: 4. Such fibronectin based scaffold proteins have improvedstability relative to fibronectin based scaffolds containing one or moreDK sequences.

In some embodiments, a fibronectin based scaffold protein comprises a¹⁰Fn3 domain having at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, or 90% identity to the human ¹⁰Fn3 domain having the amino acidsequence of SEQ ID NO: 1. Much of the variability will generally occurin one or more of the loops. Each of the beta or beta-like strands of a¹⁰Fn3 domain in a fibronectin based scaffold protein may comprise,consist essentially of, or consist of an amino acid sequence that is atleast 80%, 85%, 90%, 95% or 100% identical to the sequence of acorresponding beta or beta-like strand of SEQ ID NO: 1, provided thatsuch variation does not disrupt the stability of the polypeptide inphysiological conditions. In exemplary embodiments, the ¹⁰Fn3 domainbinds to a desired target with a K_(D) of less than 500 nM, 100 nM, 1nM, 500 pM, 100 pM or less. In exemplary embodiments, the fibronectinbased scaffold protein binds specifically to a target that is not boundby a wild-type ¹⁰Fn3 domain, particularly the wild-type human ¹⁰Fn3domain.

In some embodiments, the disclosure provides polypeptides comprising a¹⁰Fn3 domain, wherein the ¹⁰Fn3 domain comprises a loop, AB; a loop, BC;a loop, CD; a loop, DE; a loop, EF; and a loop, FG; and has at least oneloop selected from loop BC, DE, and FG with an altered amino acidsequence relative to the sequence of the corresponding loop of the human¹⁰Fn3 domain. In some embodiments, the BC and FG loops are altered. Insome embodiments, the BC, DE, and FG loops are altered, i.e., the ¹⁰Fn3domain comprises non-naturally occurring loops. By “altered” is meantone or more amino acid sequence alterations relative to a templatesequence (i.e., the corresponding human fibronectin domain) and includesamino acid additions, deletions, and substitutions. Altering an aminoacid sequence may be accomplished through intentional, blind, orspontaneous sequence variation, generally of a nucleic acid codingsequence, and may occur by any technique, for example, PCR, error-pronePCR, or chemical DNA synthesis.

In some embodiments, one or more loops selected from BC, DE, and FG maybe extended or shortened in length relative to the corresponding humanfibronectin loop. In some embodiments, the length of the loop may beextended by from 2-25 amino acids. In some embodiments, the length ofthe loop may be decreased by 1-11 amino acids. In particular, the FGloop of ¹⁰Fn3 is 12 residues long, whereas the corresponding loop inantibody heavy chains ranges from 4-28 residues. To optimize antigenbinding, therefore, the length of the FG loop of a ¹⁰Fn3 domain may bealtered in length as well as in sequence to cover the CDR3 range of 4-28residues to obtain the greatest possible flexibility and affinity inantigen binding.

In some embodiments, the fibronectin based scaffold protein comprises a¹⁰Fn3 domain having an amino acid sequence at least 80, 85, 90, 95, 98,or 100% identical to the non-loop regions of SEQ ID NO: 1, wherein atleast one loop selected from BC, DE, and FG is altered. In someembodiments, the altered BC loop has up to 10 amino acid substitutions,up to 4 amino acid deletions, up to 10 amino acid insertions, or acombination thereof. In some embodiments, the altered DE loop has up to6 amino acid substitutions, up to 4 amino acid deletions, up to 13 aminoacid insertions, or a combination thereof. In some embodiments, the FGloop has up to 12 amino acid substitutions, up to 11 amino aciddeletions, up to 25 amino acid insertions, or a combination thereof.

In certain embodiments, the fibronectin based scaffold protein comprisesa ¹⁰Fn3 domain that is defined generally by following the sequence:EVVAATPTSLLISW(X)_(x)RYYRITYGETGGNSPVQEFTVP(X)_(y)TATISGLKPGVDYTITVYAVT(X)_(z)PISINYRTEIEK (SEQ ID NO: 38)

In SEQ ID NO: 38, the BC loop is represented by X_(x), the DE loop isrepresented by X_(y), and the FG loop is represented by X_(z). Xrepresents any amino acid and the subscript following the X representsan integer of the number of amino acids. In particular, x, y and z mayeach independently be anywhere from 2-20, 2-15, 2-10, 2-8, 5-20, 5-15,5-10, 5-8, 6-20, 6-15, 6-10, 6-8, 2-7, 5-7, or 6-7 amino acids. Inpreferred embodiments, x is 7 amino acids, y is 4 amino acids, and z is6, 10 or 15 amino acids. The sequences of the beta strands (underlined)may have anywhere from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5,from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 substitutions,deletions or additions across all 7 scaffold regions relative to thecorresponding amino acids shown in SEQ ID NO: 38. In an exemplaryembodiment, the sequences of the beta strands may have anywhere from 0to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3,from 0 to 2, or from 0 to 1 conservative substitutions across all 7scaffold regions relative to the corresponding amino acids shown in SEQID NO: 38. In certain embodiments, the core amino acid residues arefixed and any substitutions, conservative substitutions, deletions oradditions occur at residues other than the core amino acid residues. TheEIEK tail (SEQ ID NO: 4) shown in bold is fixed. In certain embodiments,the amino acids immediately flanking the loop regions (e.g., thenon-underlined residues) may each independently be substituted ordeleted. When substituting the residues immediately flanking the loops,each residues may be substituted with a sequence having the same numberof amino acids or with a larger amino acid sequence (e.g., insertions of0-10, 0-8, 0-5, 0-3, or 0-2 amino acid residues). The non-underlinedresidues are part of the loop region and therefore are amenable tosubstitution without significantly affecting the structure of the ¹⁰Fn3domain.

The ¹⁰Fn3 domains generally begin with amino acid number 1 of SEQ ID NO:37. However, domains with amino acid deletions are also encompassed bythe invention. In some embodiments, the first eight amino acids of SEQID NO: 37 are deleted. Additional sequences may also be added to the N-or C-terminus of a ¹⁰Fn3 domain having amino acids corresponding to 1-94of SEQ ID NO: 37 or amino acids 9-94 of SEQ ID NO: 37. For example, anadditional MG sequence may be placed at the N-terminus of a ¹⁰Fn3domain. The M will usually be cleaved off, leaving a G at theN-terminus. In some embodiments, the N-terminal extension consists of anamino acid sequence selected from the group consisting of: M, MG, G, andany of SEQ ID NOs: 19-21.

The ¹⁰Fn3 domain may optionally comprise an N-terminal extension of from1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids in length.Exemplary N-terminal extensions include (represented by the singleletter amino acid code) M, MG, G, MGVSDVPRDL (SEQ ID NO: 19), VSDVPRDL(SEQ ID NO: 20), and GVSDVPRDL (SEQ ID NO: 21), or N-terminaltruncations of any one of SEQ ID NOs: 19, 20 or 21. Other suitableN-terminal extensions include, for example, X_(n)SDVPRDL (SEQ ID NO:26), X_(n)DVPRDL (SEQ ID NO. 27), X_(n)VPRDL (SEQ ID NO: 28), X_(n)PRDL(SEQ ID NO: 29), X_(n)RDL (SEQ ID NO: 30), X_(n)DL (SEQ ID NO: 31), orX_(n)L, wherein n=0, 1 or 2 amino acids, wherein when n=1, X is Met orGly, and when n=2, X is Met-Gly. When a Met-Gly sequence is added to theN-terminus of a ¹⁰Fn3 domain, the M will usually be cleaved off, leavinga G at the N-terminus.

The fibronectin based scaffold proteins provided herein comprise a ¹⁰Fn3domain having a C-terminal tail sequence comprising the amino acidsequence of SEQ ID NO: 4. Exemplary C-terminal tails includepolypeptides that are from 1-20, 1-15, 1-10, 1-8, 1-5, or 1-4 aminoacids in length. Specific examples of tail sequences include, forexample, polypeptides comprising, consisting essentially of, orconsisting of, EIEK (SEQ ID NO: 4), EGSGC (SEQ ID NO: 5), EIEKPCQ (SEQID NO: 6), EIEKPSQ (SEQ ID NO: 32), EIEKP (SEQ ID NO: 33), EIEKPS (SEQID NO: 34), or EIEKPC (SEQ ID NO: 35). Such C-terminal sequences arereferred to herein as tails or extensions and are further describedherein. In exemplary embodiments, the C-terminal tail comprises,consists essentially of, or consists of the amino acid sequence of SEQID NO: 6. In preferred embodiments, the C-terminal sequences lack DKsequences. In exemplary embodiments, the C-terminal tail comprises aresidue that facilitates modification by PEG, i.e., a lysine or cysteineresidue. In preferred embodiments, the C-terminal tail lacks a DKsequence and comprises a cysteine residue.

In certain embodiments, the fibronectin based scaffold proteins comprisea ¹⁰Fn3 domain having both an N-terminal extension and a C-terminaltail. In some embodiments, a His6-tag may be placed at the N-terminus orthe C-terminus.

In an exemplary embodiment, the fibronectin based scaffold proteincomprises a ¹⁰Fn3 domain that binds to VEGFR2. In certain embodiments,the fibronectin based scaffold protein binds to VEGFR2 with a K_(D) ofless than 500 nM and the BC loop comprises the amino acid sequence ofSEQ ID NO: 43, the DE comprises the amino acid sequence of SEQ ID NO:44, the FG loop comprises the amino acid sequence of SEQ ID NO: 45, andthe protein comprises a C-terminal tail that lacks a DK sequence. Inexemplary embodiments, the C-terminal tail comprises EIEK (SEQ ID NO:4), EGSGC (SEQ ID NO: 5), EIEKPCQ (SEQ ID NO: 6), EIEKPSQ (SEQ ID NO:32), EIEKP (SEQ ID NO: 33), EIEKPS (SEQ ID NO: 34), or EIEKPC (SEQ IDNO: 35). In preferred embodiments, the C-terminal tail comprises EIEK(SEQ ID NO: 4) or EIEKPCQ (SEQ ID NO: 6).

In certain embodiments, the fibronectin based scaffold protein is amultivalent protein that comprises two or more ¹⁰Fn3 domains. Forexample, a multivalent fibronectin based scaffold protein may comprise2, 3 or more ¹⁰Fn3 domains that are covalently associated. In exemplaryembodiments, the fibronectin based scaffold protein is a bispecific ordimeric protein comprising two ¹⁰Fn3 domains. In certain embodiments, amultivalent fibronectin based protein scaffold comprises a first ¹⁰Fn3domain that binds to a first target molecule and a second ¹⁰Fn3 domainthat binds to a second target molecule. The first and second targetmolecules may be the same or different target molecules. When the firstand second target molecules are the same, the ¹⁰Fn3 domains, i.e., thebinding loops, may be the same or different. Therefore, the first andsecond ¹⁰Fn3 domains may bind to the same target but at differentepitopes.

In exemplary embodiments, each ¹⁰Fn3 domain of a multivalent fibronectinbased protein scaffold binds to a desired target with a K_(D) of lessthan 500 nM, 100 nM, 1 nM, 500 pM, 100 pM or less. In exemplaryembodiments, each ¹⁰Fn3 domain of a multivalent fibronectin basedprotein scaffold binds specifically to a target that is not bound by awild-type ¹⁰Fn3 domain, particularly the wild-type human ¹⁰Fn3 domain.

The ¹⁰Fn3 domains in a multivalent fibronectin based scaffold proteinmay be connected by a polypeptide linker. Exemplary polypeptide linkersinclude polypeptides having from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3,or 1-2 amino acids. Specific examples of suitable polypeptide linkersare described further herein. In certain embodiments, the linker may bea C-terminal tail polypeptide as described herein, an N-terminalextension polypeptide as described herein, a linker polypeptide asdescribed below, or any combination thereof.

In the case of multivalent fibronectin based scaffold proteins,preferably none of the ¹⁰Fn3 domains comprise a C-terminal tailcontaining a DK sequence. In exemplary embodiments, a multivalentfibronectin based scaffold protein comprises two or more ¹⁰Fn3 domains,wherein each domain comprises a C-terminal tail that does not contain aDK sequence. In certain embodiments, a multivalent fibronectin basedscaffold protein comprises two or more ¹⁰Fn3 domains, wherein eachdomain comprises a C-terminal tail that does not contain a DK sequenceand does contain a residue suitable for addition of a PEG moiety, suchas a lysine or cysteine residue. In certain embodiments, a multivalentfibronectin based scaffold protein comprises two or more ¹⁰Fn3 domains,wherein each domain comprises a C-terminal tail comprising the aminoacid sequence of SEQ ID NO: 4. In certain embodiments, a multivalentfibronectin based scaffold protein comprises two or more ¹⁰Fn3 domains,wherein the N-terminal ¹⁰Fn3 domain comprises a C-terminal tailcomprising the amino acid sequence of SEQ ID NO: 4 and the C-terminal¹⁰Fn3 domain comprises a C-terminal tail comprising the amino acidsequence of EIEK (SEQ ID NO: 4), EGSGC (SEQ ID NO: 5), EIEKPCQ (SEQ IDNO: 6), EIEKPSQ (SEQ ID NO: 32), EIEKP (SEQ ID NO: 33), EIEKPS (SEQ IDNO: 34), or EIEKPC (SEQ ID NO: 35).

By varying the loop sequences of the 10Fn3 domain, it is possible togenerate a fibronectin based scaffold protein that binds to any desiredtarget. In exemplary embodiments, the fibronectin based scaffoldproteins provided herein having increased stability may bind to atherapeutically desirable target, such as, for example, TNFα, VEGFR2,IGF-IR, or EGFR. In certain embodiments, a fibronectin based scaffoldprotein does not bind to one or more of the following targets: EGFR,IGF-IR, HSA and PCSK9, or a fragment of any of the foregoing. In certainembodiments, a fibronectin based scaffold protein comprising a single¹⁰Fn3 domain docs not bind to one or more of the following targets:EGFR, IGF-IR, HSA and PCSK9, or a fragment of any of the foregoing. Incertain embodiments, a fibronectin based scaffold protein comprising asingle ¹⁰Fn3 domain does not bind to any of the following targets: EGFR,IGF-IR, HSA and PCSK9, or a fragment of any of the foregoing. In certainembodiments, a fibronectin based scaffold protein comprising two ¹⁰Fn3domains does not bind one or more of the following targets: EGFR,IGF-IR, HSA and PCSK9, or a fragment of any of the foregoing. In certainembodiments, a fibronectin based scaffold protein comprising two ¹⁰Fn3domains does not bind one or more of the following combinations oftarget molecules: i) EGFR and IGF-IR; ii) EGFR and any other targetprotein; or iii) human serum albumin and any other target protein. Incertain embodiments, a fibronectin based scaffold protein comprising two¹⁰Fn3 domains does not bind any of the following combinations of targetmolecules: i) EGFR and IGF-IR; ii) EGFR and any other target protein; oriii) human serum albumin and any other target protein.

Fibronectin Based Scaffold Protein Dimers

In certain embodiments, the fibronectin based scaffold proteinsdescribed herein are dimers comprising two ¹⁰Fn3 domains. In oneembodiment, the application provides V/I fibronectin based scaffoldprotein dimers comprising a first and second ¹⁰Fn3 domain selected fromthe group consisting of: (i) a ¹⁰Fn3 domain comprising a BC loop havingthe amino acid sequence of SEQ ID NO: 40, a DE loop having the aminoacid sequence of SEQ ID NO: 41, an FG loop having the amino acidsequence of SEQ ID NO: 42, and a C-terminal tail comprising the aminoacid sequence of any one of SEQ ID NOs: 4-6, wherein the ¹⁰Fn3 domainbinds IGF-IR; and (ii) a ¹⁰Fn3 domain comprising a BC loop having theamino acid sequence of SEQ ID NO: 43, a DE loop having the amino acidsequence of SEQ ID NO: 44, an FG loop having the amino acid sequence ofSEQ ID NO: 45, and a C-terminal tail comprising the amino acid sequenceof any one of SEQ ID NOs: 4-6, wherein the Fn3 domain binds VEGFR2. Inexemplary embodiments, each of the VEGFR2 and IGF-IR binding ¹⁰Fn3domains binds to its target with a K_(D) of less than 500 nM, 100 nM, 1nM, 500 pM, 100 pM or less.

In certain embodiments, the V/I fibronectin based scaffold protein dimercomprises in order from N-terminus to C-terminus an IGF-IR bindingdomain and a VEGFR2 binding domain. In exemplary embodiments, thefibronectin based scaffold protein dimer comprises: (i) a first ¹⁰Fn3domain comprising a BC loop having the amino acid sequence of SEQ ID NO:40, a DE loop having the amino acid sequence of SEQ ID NO: 41, an FGloop having the amino acid sequence of SEQ ID NO: 42, and a C-terminaltail comprising the amino acid sequence of SEQ ID NO: 4, wherein the¹⁰Fn3 domain binds IGF-IR; and (ii) a second ¹⁰Fn3 domain comprising aBC loop having the amino acid sequence of SEQ ID NO: 43, a DE loophaving the amino acid sequence of SEQ ID NO: 44, an FG loop having theamino acid sequence of SEQ ID NO: 45, and a C-terminal tail comprisingthe amino acid sequence of SEQ ID NO: 6, wherein the ¹⁰Fn3 domain bindsVEGFR2. In exemplary embodiments, each of the VEGFR2 and IGF-IR binding¹⁰Fn3 domains binds to its target with a K_(D) of less than 500 nM, 100nM, 1 nM, 500 pM, 100 pM or less.

In certain embodiments, the V/I fibronectin based scaffold protein dimercomprises in order from N-terminus to C-terminus a VEGFR2 binding domainand an IGF-IR binding domain. In exemplary embodiments, the fibronectinbased scaffold protein dimer comprises: (i) a first ¹⁰Fn3 domaincomprising a BC loop having the amino acid sequence of SEQ ID NO: 43, aDE loop having the amino acid sequence of SEQ ID NO: 44, an FG loophaving the amino acid sequence of SEQ ID NO: 45, and a C-terminal tailcomprising the amino acid sequence of SEQ ID NO: 4, wherein the ¹⁰Fn3domain binds VEGFR2; and (ii) a second ¹⁰Fn3 domain comprising a BC loophaving the amino acid sequence of SEQ ID NO: 40, a DE loop having theamino acid sequence of SEQ ID NO: 41, an FG loop having the amino acidsequence of SEQ ID NO: 42, and a C-terminal tail comprising the aminoacid sequence of SEQ ID NO: 6, wherein the ¹⁰Fn3 domain binds IGF-IR;and In exemplary embodiments, each of the VEGFR2 and IGF-IR binding¹⁰Fn3 domains binds to its target with a K_(D) of less than 500 nM, 100nM, 1 nM, 500 pM, 100 pM or less.

In certain embodiments, the V/I fibronectin based scaffold protein dimercomprises a first and second ¹⁰Fn3 domain selected from the groupconsisting of: (i) a ¹⁰Fn3 domain comprising, consisting essentially of,or consisting of an amino acid sequence having at least 90%, 95%, 97%,98%, 99% or 100% identity with SEQ ID NO: 2, wherein the ¹⁰Fn3 domaincomprises, consists essentially of, or consists of a C-terminal tailhaving the amino acid sequence of any one of SEQ ID NOs: 4-6, andwherein the ¹⁰Fn3 domain binds IGF-IR; and (ii) a ¹⁰Fn3 domaincomprising, consisting essentially of, or consisting of an amino acidsequence having at least 90%, 95%, 97%, 98%, 99% or 100% identity withSEQ ID NO: 3, wherein the ¹⁰Fn3 domain comprises, consists essentiallyof, or consists of a C-terminal tail having the amino acid sequence ofany one of SEQ ID NOs: 4-6, and wherein the ¹⁰Fn3 domain binds VEGFR2.In exemplary embodiments, each of the VEGFR2 and IGF-IR binding ¹⁰Fn3domains binds to its target with a of less than 500 nM, 100 nM, 1 nM,500 pM, 100 pM or less. In exemplary embodiments the first and second¹⁰Fn3 domains are connected via a polypeptide linker. In certainembodiments, the V/I fibronectin based scaffold protein dimer comprisesan N-terminal VEGFR2 binding ¹⁰Fn3 domain and a C-terminal IGF-IRbinding ¹⁰Fn3 domain. In certain embodiments, the V/I fibronectin basedscaffold protein dimer comprises an N-terminal IGF-IR binding ¹⁰Fn3domain and a C-terminal VEGFR2 binding ¹⁰Fn3 domain. In certainembodiments, the N-terminal ¹⁰Fn3 domain comprises a C-terminal tailhaving the amino acid sequence of SEQ ID NO: 4 and the C-terminal ¹⁰Fn3domain comprises a C-terminal tail having the amino acid sequence of SEQID NO: 6.

In certain embodiments, a V/I fibronectin based scaffold protein dimercomprises the amino acid sequence of any one of SEQ ID NOs: 48-55. Inother embodiments, a V/I fibronectin based scaffold protein dimercomprises the amino acid sequence of SEQ ID NO: 48. In some embodiments,a V/I fibronectin based scaffold protein dimer comprises an amino acidsequence having at least 70, 75, 80, 85, 90, 95, 97, 98, 99 or 100%identity with the amino acid sequence of any one of SEQ ID NOs: 48-55.

In certain embodiments, a V/T fibronectin based scaffold protein dimercomprises a polypeptide having the structure N1-D1-C1-L-N2-D2-C2,wherein N1 and N2 are optional N-terminal extensions independentlycomprising from 0-10 amino acids, wherein D1 and D2 are independentlyselected from the group consisting of: (i) a tenth fibronectin type IIIdomain (¹⁰Fn3) domain having at least 90%, 95%, 97%, 98%, 99% or 100%identity with the amino acid sequence set forth in SEQ ID NO: 2, whereinsaid ¹⁰Fn3 domain binds to IGF-IR with a K_(D) of less than 500 nM, 100nM, 1 nM, 500 pM, 100 pM or less, and (ii) a ¹⁰Fn3 domain having atleast 90%, 95%, 97%, 98%, 99% or 100% identity with the amino acidsequence set forth in SEQ ID NO: 3, wherein said ¹⁰Fn3 domain binds toVEGFR2 with a K_(D) of less than 500 nM, 100 nM, 1 nM, 500 pM, 100 pM;wherein L is a polypeptide linker comprising from 0-30 amino acidresidues; wherein C1 comprises, consists essentially of, or consists ofthe amino acid sequence of SEQ ID NO: 4; and wherein C2 comprises,consists essentially of, or consists of any one of the amino acidsequences of SEQ ID NOs: 4, 5 or 6. In certain embodiments, D1 binds toVEGFR2 and D2 binds to IGF-IR. In other embodiments, D1 binds to IGF-IRand D2 binds to VEGFR2.

In certain embodiments, the D1 or D2 region is a ¹⁰Fn3 domain that bindsto VEGFR2 comprising a BC loop having the amino acid sequence of SEQ IDNO: 43, a DE loop having the amino acid sequence of SEQ ID NO: 44, andan FG loop having the amino acid sequence of SEQ ID NO: 45, wherein the¹⁰Fn3 domain binds to VEGFR2 with a K_(D) of less than 100 nM.

In certain embodiments, the D1 or D2 region is a VEGFR2 binderrepresented by the following amino acid sequence:

(SEQ ID NO: 3) EVVAATPTSLLISW RHPHFPT RYYRITYGETGGNSPVQEFTVP LQPP TATISGLKPGVDYTITFVYAVT DGRNGRLLSI PISINYRT.In SEQ ID NO: 3, the sequence of the BC, DE and FG loops have a fixedsequence as shown in bold (e.g., a BC loop having the amino acidsequence of SEQ ID NO: 43, a DE loop having the amino acid sequence ofSEQ ID NO: 44, and an FG loop having the amino acid sequence of SEQ IDNO: 45) and the remaining sequence which is underlined (e.g., thesequence of the 7 beta strands and the AB, CD and EF loops) has anywherefrom 0 to 20, from 0 to 15, from 0 to 10, from 0 to 8, from 0 to 6, from0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1substitutions, conservative substitutions, deletions or additionsrelative to the corresponding amino acids shown in SEQ ID NO: 3. Incertain embodiments, the core amino acid residues are fixed and anysubstitutions, conservative substitutions, deletions or additions occurat residues other than the core amino acid residues.

The ¹⁰Fn3 domain that binds to VEGFR2 may optionally be linked to anN-terminal extension (N1 or N2) of from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4,1-3, 1-2, or 1 amino acids in length. Exemplary N-terminal extensionsinclude (represented by the single letter amino acid code) M, MG, G,MGVSDVPRDL (SEQ ID NO: 19), VSDVPRDL (SEQ ID NO: 20), and GVSDVPRDL (SEQID NO: 21), or N-terminal truncations of any one of SEQ ID NOs: 19, 20or 21. Other suitable N-terminal extensions include, for example,X_(n)SDVPRDL (SEQ ID NO: 26), X_(n)DVPRDL (SEQ ID NO: 27), X_(n)VPRDL(SEQ ID NO: 28), X_(n)PRDL (SEQ ID NO: 29), X_(n)RDL (SEQ ID NO: 30),X_(n)DL (SEQ ID NO: 31), or X_(n)L, wherein n=0, 1 or 2 amino acids,wherein when n=1, X is Met or Gly, and when n=2, X is Met-Gly. Inpreferred embodiments, N1 comprises, consists essentially of, orconsists of the amino acid sequence of SEQ ID NO: 19. In preferredembodiments, N2 comprises, consists essentially of, or consists of theamino acid sequence of SEQ ID NO: 20.

The ¹⁰Fn3 domain that binds to VEGFR2 may optionally comprise aC-terminal tail (C1 or C2). The C-terminal tails of the fibronectinbased scaffold protein dimers of the claimed invention do not contain aDK sequence. Exemplary C-terminal tails include polypeptides that arefrom 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids inlength. Specific examples of C-terminal tails include EIEKPSQ (SEQ IDNO: 32), EIEKPCQ (SEQ ID NO: 6), and EIEK. (SEQ ID NO: 4). In otherembodiments, suitable C-terminal tails may be a C-terminally truncatedfragment of SEQ ID NOs: 4, 6 or 32, including, for example, one of thefollowing amino acid sequences (represented by the single letter aminoacid code): EIE, EIEKP (SEQ ID NO: 33), EIEKPS (SEQ ID NO: 34), orEIEKPC (SEQ ID NO: 35). Other suitable C-terminal tails include, forexample, ES, EC, EGS, EGC, EGSGS (SEQ ID NO: 36), or EGSGC (SEQ ID NO:5). In certain embodiments. C1 comprises, consists essentially of, orconsists of the amino acid sequence of SEQ ID NO: 4. In certainembodiments, C2 comprises, consists essentially of, or consists of theamino acid sequence of SEQ ID NO: 4, 5 or 6. In preferred embodiments,C1 comprises, consists essentially of, or consists of the amino acidsequence of SEQ ID NO: 4 and C2 comprises, consists essentially of, orconsists of the amino acid sequence of SEQ ID NO: 6.

In certain embodiments, the ¹⁰Fn3 domain that binds to VEGFR2 comprisesboth an N-terminal extension and a C-terminal tail. In exemplaryembodiments, N1 begins with Gly or Met-Gly, C1 does not contain acysteine residue, N2 does not start with a Met. and C2 comprises acysteine residue. Specific examples of ¹⁰Fn3 domains that bind to VEGFR2are polypeptides comprising: (i) the amino acid sequence set forth inSEQ ID NO: 3, or (ii) an amino acid sequence having at least 85%, 90%,95%, 97%, 98%, or 99% identity with the amino acid sequence set forth inSEQ ID NO: 3.

In certain embodiments, the D1 or D2 region is a ¹⁰Fn3 domain that bindsto IGF-IR comprising a BC loop having the amino acid sequence of SEQ IDNO: 40, a DE loop having the amino acid sequence of SEQ ID NO: 41, andan FG loop having the amino acid sequence of SEQ ID NO: 42, wherein the¹⁰Fn3 domain binds to IGF-IR with a K_(D) of less than 100 nM.

In certain embodiments, the D1 or D2 region is an IGF-IR binderrepresented by the following amino acid sequence:

(SEQ ID NO: 2) EVVAATPTSLLISW SARLKVA RYYRITYGETGGNSPVQEFTVP KNVY TATISGLKPGVDYTITVYAVT RFRQYQ PISINYRT.

In SEQ ID NO: 2, the sequence of the BC, DE and FG loops have a fixedsequence as shown in bold (e.g., a BC loop having the amino acidsequence of SEQ ID NO: 40, a DE loop having the amino acid sequence ofSEQ ID NO: 41, and an FG loop having the amino acid sequence of SEQ IDNO: 42) and the remaining sequence which is underlined (e.g., thesequence of the 7 beta strands and the AB, CD and EF loops) has anywherefrom 0 to 20, from 0 to 15, from 0 to 10, from 0 to 8, from 0 to 6, from0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1substitutions, conservative substitutions, deletions or additionsrelative to the corresponding amino acids shown in SEQ ID NO: 2. Incertain embodiments, the core amino acid residues are fixed and anysubstitutions, conservative substitutions, deletions or additions occurat residues other than the core amino acid residues.

The ¹⁰Fn3 domain that binds to IGF-IR may optionally be linked to anN-terminal extension (N1 or N2) of from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4,1-3, 1-2, or 1 amino acids in length. Exemplary N-terminal extensionsinclude (represented by the single letter amino acid code) M, MG, G,MGVSDVPRDL (SEQ ID NO: 19), VSDVPRDL (SEQ ID NO: 20), and GVSDVPRDL (SEQID NO: 21), or N-terminal truncations of any one of SEQ ID NOs: 19, 20or 21. Other suitable N-terminal extensions include, for example,X_(n)SDVPRDL (SEQ ID NO: 26), X_(n)DVPRDL (SEQ ID NO: 27), X_(n)VPRDL(SEQ ID NO: 28), X_(n)PRDL (SEQ ID NO: 29), X_(n)RDL (SEQ ID NO: 30),X_(n)DL (SEQ ID NO: 31), or X_(n)L, wherein n=0, 1 or 2 amino acids,wherein when n=1, X is Met or Gly, and when n=2, X is Met-Gly. Inpreferred embodiments, N1 comprises, consists essentially of, orconsists of the amino acid sequence of SEQ ID NO: 19. In preferredembodiments, N2 comprises, consists essentially of, or consists of theamino acid sequence of SEQ ID NO: 20.

The ¹⁰Fn3 domain that binds to IGF-IR may optionally comprise aC-terminal tail (C1 or C2). The C-terminal tails of the fibronectinbased scaffold protein dimers of the claimed invention do not contain aDK sequence. Exemplary C-terminal tails include polypeptides that arefrom 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids inlength. Specific examples of C-terminal tails include EIEKPSQ (SEQ IDNO: 32), EIEKPCQ (SEQ ID NO: 6), and EIEK (SEQ ID NO: 4). In otherembodiments, suitable C-terminal tails may be a C-terminally truncatedfragment of SEQ ID NOs: 4, 6 or 32, including, for example, one of thefollowing amino acid sequences (represented by the single letter aminoacid code): EIE, EIEKP (SEQ ID NO: 33), EIEKPS (SEQ ID NO: 34), orEIEKPC (SEQ ID NO: 35). Other suitable C-terminal tails include, forexample, ES, EC, EGS, EGC, EGSGS (SEQ ID NO: 36), or EGSGC (SEQ ID NO:5). In certain embodiments, C1 comprises, consists essentially of, orconsists of the amino acid sequence of SEQ ID NO: 4. In certainembodiments, C2 comprises, consists essentially of, or consists of theamino acid sequence of SEQ ID NO: 4, 5 or 6. In preferred embodiments.C1 comprises, consists essentially of, or consists of the amino acidsequence of SEQ ID NO: 4 and C2 comprises, consists essentially of, orconsists of the amino acid sequence of SEQ ID NO: 6.

In certain embodiments, the ¹⁰Fn3 domain that binds to IGF-IR comprisesboth an N-terminal extension and a C-terminal tail. In exemplaryembodiments, N1 begins with Gly or Met-Gly, C1 does not contain acysteine residue, N2 does not start with a Met, and C2 comprises acysteine residue. Specific examples of ¹⁰Fn3 domains that bind to IGF-IRare polypeptides comprising: (i) the amino acid sequence set forth inSEQ ID NO: 2, or (ii) an amino acid sequence having at least 85%, 90%,95%, 97%, 98%, or 99% identity with the amino acid sequence set forth inSEQ ID NO: 2.

The L region is a polypeptide linker. Exemplary polypeptide linkersinclude polypeptides having from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3,or 1-2 amino acids. Specific examples of suitable polypeptide linkersare described further herein. In certain embodiments, the linker may bea C-terminal tail polypeptide as described herein, an N-terminalextension polypeptide as described herein, or a combination thereof.

In certain embodiments, one or more of N1, N2, L, C1 or C2 may comprisean amino acid residue suitable for pegylation, such as a cysteine orlysine residue. In exemplary embodiments, C2 comprises at least oneamino acid suitable for pegylation, such as a cysteine or lysineresidue. Specific examples of suitable polypeptide linkers are describedfurther below. Specific examples of fibronectin based scaffold proteindimers having the structure N1-D1-C1-L-N2-D2-C2 are polypeptidescomprising: (i) the amino acid sequence set forth in any one of SEQ IDNOs: 48-55, or (ii) an amino acid sequence having at least 85%, 90%,95%, 97%, 98%, or 99% identity with any one of SEQ ID NOs: 48-55.

In certain embodiments, fibronectin based scaffold protein dimers willhave the structure N1-D1-L-N2-D2, wherein D1 and D2 each comprise anamino acid sequence having the following sequence:

(SEQ ID NO: 38) EVVAATPTSLLISW(X)_(x)RYYRITYGETGGNSPVQEFTVP(X)_(y)TATISGLKPGVDYTITVYAVT(X)_(z)PISINYRT EIEKIn SEQ ID NO: 38, the BC loop is represented by X_(x), the DE loop isrepresented by X_(y), and the FG loop is represented by X_(z). Thesequences of the beta strands (underlined) may have anywhere from 0 to10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3,from 0 to 2, or from 0 to 1 substitutions, deletions or additions acrossall 7 scaffold regions relative to the corresponding amino acids shownin SEQ ID NO: 38. In an exemplary embodiment, the sequences of the betastrands may have anywhere from 0 to 10, from 0 to 8, from 0 to 6, from 0to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 conservativesubstitutions across all 7 scaffold regions relative to thecorresponding amino acids shown in SEQ ID NO: 38. In certainembodiments, the core amino acid residues are fixed and anysubstitutions, conservative substitutions, deletions or additions occurat residues other than the core amino acid residues. The EIEK tail (SEQID NO: 4) shown in bold is fixed. In certain embodiments, the aminoacids immediately flanking the loop regions (e.g., the non-underlinedresidues) may each independently be substituted or deleted. Whensubstituting the residues immediately flanking the loops, each residuesmay be substituted with a sequence having the same number of amino acidsor with a larger amino acid sequence (e.g., insertions of 0-10, 0-8,0-5, 0-3, or 0-2 amino acid residues). The non-underlined residues arepart of the loop region and therefore are amenable to substitutionwithout significantly affecting the structure of the ¹⁰Fn3 domain.

In certain embodiments, a fibronectin based scaffold protein dimer hasthe structure N1-D1-L-N2-D2, wherein D1 and D2 are selected from thegroup consisting of: (i) a ¹⁰Fn3 domain comprising SEQ ID NO: 38,wherein X_(x) comprises, consists essentially of, or consists of theamino acid sequence of SEQ ID NO: 40, X_(y) comprises, consistsessentially of, or consists of the amino acid sequence of SEQ ID NO: 41,and X_(z) comprises, consists essentially of, or consists of the aminoacid sequence of SEQ ID NO: 42; and (ii) a ¹⁰Fn3 domain comprising SEQID NO: 38, wherein X_(x) comprises, consists essentially of, or consistsof the amino acid sequence of SEQ ID NO: 43, X_(y) comprises, consistsessentially of, or consists of the amino acid sequence of SEQ ID NO: 44,and X_(z) comprises, consists essentially of, or consists of the aminoacid sequence of SEQ ID NO: 45.

In an exemplary embodiment, a fibronectin based scaffold protein dimerhas the structure N1-D1-L-N2-D2, wherein D1 comprises the amino acidsequence of SEQ ID NO: 38, wherein X_(x) comprises, consists essentiallyof, or consists of the amino acid sequence of SEQ ID NO: 40, X_(y)comprises, consists essentially of, or consists of the amino acidsequence of SEQ ID NO: 41, and X_(z) comprises, consists essentially of,or consists of the amino acid sequence of SEQ ID NO: 42, and wherein D2comprises the amino acid sequence of SEQ ID NO: 38, wherein X_(x)comprises, consists essentially of, or consists of the amino acidsequence of SEQ ID NO: 43, X_(y) comprises, consists essentially of, orconsists of the amino acid sequence of SEQ ID NO: 44, and X_(z)comprises, consists essentially of, or consists of the amino acidsequence of SEQ ID NO: 45.

In another exemplary embodiment, a fibronectin based scaffold proteindimer has the structure N1-D1-L-N2-D2, wherein D1 comprises the aminoacid sequence of SEQ ID NO: 38, wherein X_(x) comprises, consistsessentially of, or consists of the amino acid sequence of SEQ ID NO: 43,X_(y) comprises, consists essentially of, or consists of the amino acidsequence of SEQ ID NO: 44, and X_(z) comprises, consists essentially of,or consists of the amino acid sequence of SEQ ID NO: 45; and wherein D2comprises the amino acid sequence of SEQ ID NO: 38, wherein X_(x)comprises, consists essentially of, or consists of the amino acidsequence of SEQ ID NO: 40, X_(y) comprises, consists essentially of, orconsists of the amino acid sequence of SEQ ID NO: 41, and X_(z)comprises, consists essentially of, or consists of the amino acidsequence of SEQ ID NO: 42.

In certain embodiments, a fibronectin based scaffold protein dimerhaving the structure N1-D1-L-N2-D2 further comprises a C-terminal tail.In exemplary embodiments, the C-terminal tail comprises a residuesuitable for addition of a PEG moiety, e.g., a lysine or cysteineresidue. In a preferred embodiment, the C-terminal tail comprises thesequence PCQ.

In various embodiments, the L domain of a fibronectin based scaffoldprotein dimer having the structure N1-D1-L-N2-D2 is a polypeptidelinker. Exemplary polypeptide linkers include polypeptides having from1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, or 1-2 amino acids. Specificexamples of suitable polypeptide linkers are described further herein.In addition, N1 and N2 are N-terminal extensions as described hereinabove.

In preferred embodiments, the fibronectin based scaffold protein dimersdescribed herein have increased stability either in vitro, in vivo orboth. In certain embodiments, the fibronectin based scaffold proteindimers described herein have reduced fragmentation and/or decreasedaggregation during storage in solution. In certain embodiments, thefibronectin based scaffold protein dimers described herein haveincreased serum half-life.

In exemplary embodiments, the fibronectin based scaffold protein dimersdescribed herein have reduced fragmentation relative to a fibronectinbased scaffold protein dimer comprising a DK sequence. In particular,the fibronectin based scaffold protein dimers described herein haveincreased stability relative to a fibronectin based scaffold proteindimer having one or more DK sequences in any one of: a C-terminal tail,an N-terminal extension or a linker between two ¹⁰Fn3 domains. Forexample, the fibronectin based scaffold protein dimers are generallymore stable than fibronectin based scaffold protein dimers having a DKsequence in one or both C-terminal tail regions, e.g., comprising a tailhaving SEQ ID NO: 46 after the first and/or second ¹⁰Fn3 subunit. Inexemplary embodiments, the fibronectin based scaffold protein dimersdescribed herein have reduced fragmentation relative to a fibronectinbased scaffold protein dimer having the formula N1-D1-C1-L-N2-D2-C2,wherein C1 and/or C2 comprise SEQ ID NO: 46. Fragmentation may beassessed, for example, using RP-HPLC analysis, as described in Example3.

In exemplary embodiments, the fibronectin based scaffold protein dimersdescribed herein exhibit less than 7%, 6%, 5%, 4%, 3.5%, 3%, 2% or lessfragmentation upon storage in solution for four weeks at pH 4.0. Incertain embodiments, the fibronectin based scaffold protein dimersdescribed herein exhibit a level of fragmentation that is reduced by atleast 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or more relative to anequivalent version of the fibronectin based scaffold protein dimer thatcontains one or more DK sequences.

In exemplary embodiments, the fibronectin based scaffold protein dimersdescribed herein exhibit a serum half-life that is increased by at least10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or more relative to theserum half-life of a an equivalent version of the fibronectin basedscaffold protein dimer that contains one or more DK sequences. In otherembodiments, the fibronectin based scaffold protein dimers describedherein exhibit a serum half-life that is increased by at least 2-fold,3-fold, 5-fold, 10-fold, or more relative to the serum half-life of a anequivalent version of the fibronectin based scaffold protein dimer thatcontains one or more DK sequences.

In certain embodiments, the application provides an E/I fibronectinbased scaffold protein dimer, comprising one ¹⁰Fn3 domain that binds toEGFR and one ¹⁰Fn3 domain that binds to IGF-IR. In certain embodiments,an E/I fibronectin based scaffold protein dimer comprises the amino acidsequence of any one of SEQ ID NOs: 22-25. In other embodiments, an E/Ifibronectin based scaffold protein dimer comprises the amino acidsequence of SEQ ID NO: 25. In some embodiments, an E/I fibronectin basedscaffold protein dimer comprises an amino acid sequence having at least70, 75, 80, 85, 90, 95, 97, 98, 99 or 100% identity with the amino acidsequence of any one of SEQ ID NOs: 22-25.

Polypeptide Linkers

The application provides multivalent fibronectin based scaffold proteinscomprising at least two ¹⁰Fn3 domains linked via a polypeptide linker.In one embodiment, the application provides fibronectin based scaffolddimers comprising two ¹⁰Fn3 domains linked via a polypeptide linker (L).The polypeptides comprise an N-terminal domain comprising a first ¹⁰Fn3domain and a C-terminal domain comprising a second ¹⁰Fn3 domain. Thefirst and second ¹⁰Fn3 domains may be directly or indirectly linked viaa polypeptide linker (L). Additional linkers or spacers, e.g., SEQ IDNOS: 4, 6 or 32, may be introduced at the C-terminus of the first ¹⁰Fn3domain between the ¹⁰Fn3 domain and the polypeptide linker. Additionallinkers or spacers may be introduced at the N-terminus of the second¹⁰Fn3 domain between the ¹⁰Fn3 domain and the polypeptide linker.

Suitable linkers for joining the ¹⁰Fn3 domains are those which allow theseparate domains to fold independently of each other forming a threedimensional structure that permits high affinity binding to a targetmolecule. The application provides that suitable linkers that meet theserequirements comprise glycine-serine based linkers, glycine-prolinebased linkers, proline-alanine based linkers as well as the linker SEQID NO: 7. The Examples described in WO 2009/142773 demonstrate that Fn3domains joined via these linkers retain their target binding function.In some embodiments, the linker is a glycine-serine based linker. Theselinkers comprise glycine and serine residues and may be between 8 and50, 10 and 30, and 10 and 20 amino acids in length. Examples of suchlinkers include SEQ ID NOs: 8-12. In some embodiments the polypeptidelinker is selected from SEQ ID NOs: 8 and 9. In some embodiments, thelinker is a glycine-proline based linker. These linkers comprise glycineand proline residues and may be between 3 and 30, 10 and 30, and 3 and20 amino acids in length. Examples of such linkers include SEQ ID NOs:13, 14 and IS. In some embodiments, the linker is a proline-alaninebased linker. These linkers comprise proline and alanine residues andmay be between 3 and 30, 10 and 30, 3 and 20 and 6 and 18 amino acids inlength. Examples of such linkers include SEQ ID NOs: 16, 17 and 18. Itis contemplated, that the optimal linker length and amino acidcomposition may be determined by routine experimentation by methods wellknown in the art. In some embodiments, the polypeptide linker is SEQ IDNO: 7. In exemplary embodiments, the linker does not contain any DKsequences.

Pharmacokinetic Moieties

In one aspect, the application provides for fibronectin based scaffoldproteins further comprising a pharmacokinetic (PK) moiety.Pharmokinetics encompasses properties of a compound including, by way ofexample, absorption, distribution, metabolism, and elimination by asubject. Improved pharmacokinetics may be assessed according to theperceived therapeutic need. Often it is desirable to increasebioavailability and/or increase the time between doses, possibly byincreasing the time that a protein remains available in the serum afterdosing. In some instances, it is desirable to improve the continuity ofthe serum concentration of the protein over time (e.g., decrease thedifference in serum concentration of the protein shortly afteradministration and shortly before the next administration). Thefibronectin based scaffold proteins may be attached to a moiety thatreduces the clearance rate of the polypeptide in a mammal (e.g., mouse,rat, or human) by greater than three-fold relative to the unmodifiedpolypeptide. Other measures of improved pharmacokinetics may includeserum half-life, which is often divided into an alpha phase and a betaphase. Either or both phases may be improved significantly by additionof an appropriate moiety. A PK moiety refers to any protein, peptide, ormoiety that affects the pharmacokinetic properties of a biologicallyactive molecule when fused to the biologically active molecule.

PK moieties that tend to slow clearance of a protein from the bloodinclude polyoxyalkylene moieties, e.g., polyethylene glycol, sugars(e.g., sialic acid), and well-tolerated protein moieties (e.g., Fc, Fcfragments, transferrin, or serum albumin). The fibronectin basedscaffold proteins may be fused to albumin or a fragment (portion) orvariant of albumin as described in U.S. Publication No. 20070048282. Insome embodiments, the PK moiety is a serum albumin binding protein suchas those described in U.S. Publication Nos. 2007/0178082 and2007/0269422. In some embodiments, the PK moiety is a serumimmunoglobulin binding protein such as those described in U.S.Publication No. 2007/0178082.

In some embodiments, the fibronectin based scaffold proteins may beattached to a PK moiety comprising a nonproteinaceous polymer. In someembodiments, the polymer is polyethylene glycol (“PEG”), polypropyleneglycol, or polyoxyalkylenes, as described in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. In exemplaryembodiments, the polymer is a PEG moiety.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Theterm “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented by the formula; X—O(CH₂CH₂O)_(n-1)CH₂CH₂OH(1), where n is 20 to 2300 and X is H or a terminal modification, e.g.,a CM alkyl. In one embodiment, the PEG of the invention terminates onone end with hydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). APEG can contain further chemical groups which are necessary for bindingreactions; which results from the chemical synthesis of the molecule; orwhich is a spacer for optimal distance of parts of the molecule. Inaddition, such a PEG can consist of one or more PEG side-chains whichare linked together. PEGs with more than one PEG chain are calledmultiarmed or branched PEGs. Branched PEGs can be prepared, for example,by the addition of polyethylene oxide to various polyols, includingglycerol, pentaerythriol, and sorbitol. For example, a four-armedbranched PEG can be prepared from pentaerythriol and ethylene oxide.Branched PEG are described in, for example, European PublishedApplication No. 473084A and U.S. Pat. No. 5,932,462. One form of PEGsincludes two PEG side-chains (PEG2) linked via the primary amino groupsof a lysine (Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62-69).

PEG conjugation to peptides or proteins generally involves theactivation of PEG and coupling of the activated PEG-intermediatesdirectly to target proteins/peptides or to a linker, which issubsequently activated and coupled to target proteins/peptides (seeAbuchowski, A. et al, J. Biol. Chem., 252, 3571 (1977) and J. Biol.Chem., 252, 3582 (1977), Zalipsky, et al., and Harris et. al., in:Poly(ethylene glycol) Chemistry: Biotechnical and BiomedicalApplications; (J. M. Harris ed.) Plenum Press: New York, 1992; Chap. 21and 22). It is noted that a fibronectin based scaffold proteincontaining a PEG molecule is also known as a conjugated protein, whereasthe protein lacking an attached PEG molecule can be referred to asunconjugated.

The size of PEG utilized will depend on several factors including theintended use of the fibronectin based scaffold protein. Larger PEGs arepreferred to increase half life in the body, blood, non-bloodextracellular fluids or tissues. For in vivo cellular activity, PEGs ofthe range of about 10 to 60 kDa are preferred, as well as PEGs less thanabout 100 kDa and more preferably less than about 60 kDa, though sizesgreater than about 100 kDa can be used as well. For in vivo imagingapplications, smaller PEGs, generally less than about 20 kDa, may beused that do not increase half life as much as larger PEGs so as topermit quicker distribution and less half life. A variety of molecularmass forms of PEG can be selected, e.g., from about 1,000 Daltons (Da)to 100,000 Da (n is 20 to 2300), for conjugating to fibronectin basedscaffold proteins. The number of repeating units “n” in the PEG isapproximated for the molecular mass described in Daltons. It ispreferred that the combined molecular mass of PEG on an activated linkeris suitable for pharmaceutical use. Thus, in one embodiment, themolecular mass of the PEG molecules does not exceed 100,000 Da. Forexample, if three PEG molecules are attached to a linker, where each PEGmolecule has the same molecular mass of 12,000 Da (each n is about 270),then the total molecular mass of PEG on the linker is about 36,000 Da(total n is about 820). The molecular masses of the PEG attached to thelinker can also be different, e.g., of three molecules on a linker twoPEG molecules can be 5,000 Da each (each n is about 110) and one PEGmolecule can be 12,000 Da (n is about 270). In some embodiments, one PEGmoiety is conjugated to the fibronectin based scaffold protein. In someembodiments, the PEG moiety is about 20, 30, 40, 50, 60, 70, 80, or 90KDa. In some embodiments, the PEG moiety is about 40 KDa.

In some embodiments, PEGylated fibronectin based scaffold proteinscontain one, two or more PEG moieties. In one embodiment, the PEGmoiety(ies) are bound to an amino acid residue which is on the surfaceof the protein and/or away from the surface that contacts the targetligand. In one embodiment, the combined or total molecular mass of PEGin a pegylated fibronectin based scaffold protein is from about 3,000 Dato 60,000 Da, or from about 10,000 Da to 36,000 Da. In a one embodiment,the PEG in a pegylated fibronectin based scaffold protein is asubstantially linear, straight-chain PEG.

One skilled in the art can select a suitable molecular mass for PEG,e.g., based on how the pegylated fibronectin based scaffold protein willbe used therapeutically, the desired dosage, circulation time,resistance to proteolysis, immunogenicity, and other considerations. Fora discussion of PEG and its use to enhance the properties of proteins,see N. V. Katre, Advanced Drug Delivery Reviews 10: 91-114 (1993).

In some embodiments, a fibronectin based scaffold protein is covalentlylinked to one poly(ethylene glycol) group of the formula:—CO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR, with the —CO (i.e. carbonyl) of thepoly(ethylene glycol) group forming an amide bond with one of the aminogroups of the binding polypeptide; R being lower alkyl; x being 2 or 3;m being from about 450 to about 950; and n and m being chosen so thatthe molecular weight of the conjugate minus the binding polypeptide isfrom about 10 to 40 kDa. In one embodiment, a fibronectin based scaffoldprotein's ε-amino group of a lysine is the available (free) amino group.

In one specific embodiment, carbonate esters of PEG are used to form thePEG-fibronectin based scaffold protein conjugates.N,N′-disuccinimidylcarbonate (DSC) may be used in the reaction with PEGto form active mixed PEG-succinimidyl carbonate that may be subsequentlyreacted with a nucleophilic group of a linker or an amino group of afibronectin based scaffold protein (see U.S. Pat. Nos. 5,281,698 and5,932,462). In a similar type of reaction,1,1′-(dibenzotriazolyl)carbonate and di-(2-pyridyl)carbonate may bereacted with PEG to form PEG-benzotriazolyl and PEG-pyridyl mixedcarbonate (U.S. Pat. No. 5,382,657), respectively.

Pegylation of a fibronectin based scaffold protein can be performedaccording to the methods of the state of the art, for example byreaction of the fibronectin based scaffold protein withelectrophilically active PEGs (supplier: Shearwater Corp., USA, worldwide web at shearwatercorp.com). Preferred PEG reagents of the presentinvention are, e.g., N-hydroxysuccinimidyl propionates (PEG-SPA),butanoates (PEG-SBA), PEG-succinimidyl propionate or branchedN-hydroxysuccinimides such as mPEG2-NHS (Monfardini, C., et al.,Bioconjugate Chem. 6 (1995) 62-69). Such methods may used to pegylate atan ε-amino group of a lysine of a fibronectin based scaffold protein orat the N-terminal amino group of the fibronectin based scaffold protein.

In another embodiment, PEG molecules may be coupled to sulfhydryl groupson a fibronectin based scaffold protein (Sartore, L., et al., Appl.Biochem. Biotechnol., 27, 45 (1991); Morpurgo et al., Biocon. Chem., 7,363-368 (1996); Goodson et al., Bio/Technology (1990) 8, 343; U.S. Pat.No. 5,766,897). U.S. Pat. Nos. 6,610,281 and 5,766,897 describesexemplary reactive PEG species that may be coupled to sulfhydryl groups.

In some embodiments, the pegylated fibronectin based scaffold protein isproduced by site-directed pegylation, particularly by conjugation of PEGto a cysteine moiety. In certain embodiments, the Cys residue may bepositioned at the N-terminus, between the N-terminus and the mostN-terminal beta or beta-like strand, at the C-terminus, or between theC-terminus and the most C-terminal beta or beta-like strand of thefibronectin based scaffold protein. In certain embodiments, thefibronectin based scaffold protein is a dimer and the Cys residue may bepositioned at the N-terminus, between the N-terminus and the mostN-terminal beta or beta-like strand, at the C-terminus, or between theC-terminus and the most C-terminal beta or beta-like strand of eitherbinding domain of the fibronectin based scaffold protein dimer. Incertain embodiments, the Cys residue may be positioned at the N-terminusof the fibronectin based scaffold protein dimer, between the N-terminusand the most N-terminal beta or beta-like strand of the fibronectinbased scaffold protein dimer (i.e., of the N-terminal binding domain ofthe fibronectin based scaffold protein dimer), or at the C-terminus ofthe fibronectin based scaffold protein dimer, or between the C-terminusand the most C-terminal beta or beta-like strand of the fibronectinbased scaffold protein dimer (i.e., of the C-terminal binding domain ofthe fibronectin based scaffold protein dimer). A Cys residue may besituated at other positions as well, particularly any of the loops thatdo not participate in target binding or between two binding domains of amultivalent fibronectin based scaffold protein. A PEG moiety may also beattached by other chemistry, including by conjugation to amines.

In some embodiments where PEG molecules are conjugated to cysteineresidues on a fibronectin based scaffold protein, the cysteine residuesare native to the fibronectin based scaffold protein, whereas in otherembodiments, one or more cysteine residues are engineered into thefibronectin based scaffold protein. Mutations may be introduced into afibronectin based scaffold protein coding sequence to generate cysteineresidues. This might be achieved, for example, by mutating one or moreamino acid residues to cysteine. Preferred amino acids for mutating to acysteine residue include serine, threonine, alanine and otherhydrophilic residues. Preferably, the residue to be mutated to cysteineis a surface-exposed residue. Algorithms are well-known in the art forpredicting surface accessibility of residues based on primary sequenceor a protein. Alternatively, surface residues may be predicted bycomparing the amino acid sequences of fibronectin based scaffoldproteins, given that the crystal structure of the tenth fn3 domainframework based on which fibronectin based scaffold proteins aredesigned has been solved (see Dickinson, et al., J. Mol. Biol. 236(4):1079-92 (1994)) and thus the surface-exposed residues identified. In oneembodiment, cysteine residues are introduced into fibronectin basedscaffold protein at or near the N- and/or C-terminus, or within loopregions. Pegylation of cysteine residues may be carried out using, forexample, PEG-maleimide, PEG-vinylsulfone, PEG-iodoacetamide, orPEG-orthopyridyl disulfide.

In some embodiments, the pegylated fibronectin based scaffold proteincomprises a PEG molecule covalently attached to the alpha amino group ofthe N-terminal amino acid. Site specific N-terminal reductive animationis described in Pepinsky et al., (2001) JPET, 297,1059, and U.S. Pat.No. 5,824,784. The use of a PEG-aldehyde for the reductive animation ofa protein utilizing other available nucleophilic amino groups isdescribed in U.S. Pat. No. 4,002,531, in Wieder et al., (1979) J. Biol.Chem. 254,12579, and in Chamow et al., (1994) Bioconjugate Chem. 5, 133.

In another embodiment, pegylated fibronectin based scaffold proteinscomprise one or more PEG molecules covalently attached to a linker,which in turn is attached to the alpha amino group of the amino acidresidue at the N-terminus of the fibronectin based scaffold protein.Such an approach is disclosed in U.S. Publication No. 2002/0044921 andPCT Publication No.

In one embodiment, a fibronectin based scaffold protein is pegylated atthe C-terminus. In a specific embodiment, a protein is pegylated at theC-terminus by the introduction of C-terminal azido-methionine and thesubsequent conjugation of a methyl-PEG-triarylphosphine compound via theStaudinger reaction. This C-terminal conjugation method is described inCazalis et al., C-Terminal Site-Specific PEGylation of a TruncatedThrombomodulin Mutant with Retention of Full Bioactivity, BioconjugChem. 2004; 15(5): 1005-1009.

In exemplary embodiments, a fibronectin based scaffold protein ispegylated in a C-terminal tail region as described further herein.Exemplary C-terminal tails include, for example, a polypeptide havingany one of SEQ ID NOs: 5, 6 or 35.

Conventional separation and purification techniques known in the art canbe used to purify PEGylated fibronectin based scaffold proteins, such assize exclusion (e.g., gel filtration) and ion exchange chromatography.Products may also be separated using SDS-PAGE. Products that may beseparated include mono-, di-, tri- poly- and un-pegylated fibronectinbased scaffold proteins, as well as free PEG. The percentage of mono-PEGconjugates can be controlled by pooling broader fractions around theelution peak to increase the percentage of mono-PEG in the composition.About ninety percent mono-PEG conjugates represents a good balance ofyield and activity. Compositions in which, for example, at leastninety-two percent or at least ninety-six percent of the conjugates aremono-PEG species may be desired. In an embodiment of this invention thepercentage of mono-PEG conjugates is from ninety percent to ninety-sixpercent.

In one embodiment of the invention, the PEG in a pegylated fibronectinbased scaffold protein is not hydrolyzed from the pegylated amino acidresidue using a hydroxylamine assay, e.g., 450 mM hydroxylamine (pH 6.5)over 8 to 16 hours at room temperature, and is thus stable. In oneembodiment, greater than 80% of the composition is stablemono-PEG-fibronectin based scaffold protein, more preferably at least90%, and most preferably at least 95%.

In another embodiment, the pegylated fibronectin based scaffold proteinswill preferably retain at least about 25%, 50%, 60%, 70%, 80%, 85%, 90%,95% or 100% of the biological activity associated with the unmodifiedprotein. In one embodiment, biological activity refers to its ability tobind to one or more target molecules, as assessed by K_(D), k_(on) ork_(off). In one specific embodiment, the pegylated fibronectin basedscaffold protein shows an increase in binding to one or more targetmolecules relative to unpegylated fibronectin based scaffold protein.

The serum clearance rate of PEG-modified fibronectin based scaffoldproteins may be decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, or even 90%, relative to the clearance rate of the unmodifiedfibronectin based scaffold protein. The PEG-modified fibronectin basedscaffold protein may have a half-life (t_(1/2)) which is enhancedrelative to the half-life of the unmodified fibronectin based scaffoldprotein. The half-life of PEG-modified fibronectin based scaffoldprotein may be enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, oreven by 1000% relative to the half-life of the unmodified fibronectinbased scaffold protein. In some embodiments, the protein half-life isdetermined in vitro, such as in a buffered saline solution or in serum.In other embodiments, the protein half-life is an in vivo half life,such as the half-life of the fibronectin based scaffold protein in theserum or other bodily fluid of an animal.

Nucleic Acid-Protein Fusion Technology

In one aspect, the application provides fibronectin based scaffoldproteins comprising a fibronectin type III domain that bind a humantarget, such as, for example, TNF-alpha, EGFR, VEGFR2, IGF-IR, or otherproteins. One way to rapidly make and test Fn3 domains with specificbinding properties is the nucleic acid-protein fusion technology ofAdnexus, a Bristol-Myers Squibb Company. Such in vitro expression andtagging technology, termed PROfusion™, that exploits nucleicacid-protein fusions (RNA- and DNA-protein fusions) may be used toidentify novel polypeptides and amino acid motifs that are important forbinding to proteins. Nucleic acid-protein fusion technology is atechnology that covalently couples a protein to its encoding geneticinformation. For a detailed description of the RNA-protein fusiontechnology and fibronectin-based scaffold protein library screeningmethods see Szostak et al., U.S. Pat. Nos. 6,258,558; 6,261,804;6,214,553; 6,281,344; 6,207,446; 6,518,018; PCT Publication Nos.WO00/34784; WO01/64942; WO02/032925; and Roberts and Szostak, Proc Natl.Acad. Sci. 94:12297-12302, 1997, herein incorporated by reference.

Vectors & Polynucleotides

Nucleic acids encoding any of the various fibronectin based scaffoldproteins disclosed herein may be synthesized chemically, enzymaticallyor recombinantly. Codon usage may be selected so as to improveexpression in a cell. Such codon usage will depend on the cell typeselected. Specialized codon usage patterns have been developed for E.coli and other bacteria, as well as mammalian cells, plant cells, yeastcells and insect cells. See for example: Mayfield et al., Proc Natl AcadSci USA. 2003 Jan. 21; 100(2):438-42; Sinclair et al. Protein ExprPurif. 2002 October; 26(1):96-105; Connell N D. Curr Opin Biotechnol.2001 October; 12(5):446-9; Makrides et al. Microbiol Rev. 1996September; 60(3):512-38; and Sharp et al. Yeast. 1991 October;7(7):657-78.

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual,Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F.Ausubel et al., Current Protocols in Molecular Biology (Green Publishingand Wiley-Interscience: New York, 1987) and periodic updates, hereinincorporated by reference. The DNA encoding the polypeptide is operablylinked to suitable transcriptional or translational regulatory elementsderived from mammalian, viral, or insect genes. Such regulatory elementsinclude a transcriptional promoter, an optional operator sequence tocontrol transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants are additionally incorporated.

The fibronectin based scaffold proteins described herein may be producedrecombinantly not only directly, but also as a fusion polypeptide with aheterologous polypeptide, which is preferably a signal sequence or otherpolypeptide having a specific cleavage site at the N-terminus of themature protein or polypeptide. The heterologous signal sequence selectedpreferably is one that is recognized and processed (i.e., cleaved by asignal peptidase) by the host cell. For prokaryotic host cells that donot recognize and process a native signal sequence, the signal sequenceis substituted by a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,a factor leader (including Saccharomyces and Kluyveromyces alpha-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or the signal described in PCT Publication No. WO90/13646. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable. The DNA for such precursor regions may be ligated in readingframe to DNA encoding the protein.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2 micron plasmid origin is suitablefor yeast, and various viral origins (SV40, polyoma, adenovirus, VSV orBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

A suitable selection gene for use in yeast is the trp 1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the fibronectin-based scaffold protein. Promoters suitablefor use with prokaryotic hosts include the phoA promoter, beta-lactamaseand lactose promoter systems, alkaline phosphatase, a tryptophan (trp)promoter system, and hybrid promoters such as the tac promoter. However,other known bacterial promoters are suitable. Promoters for use inbacterial systems also will contain a Shine-Dalgarno (S.D.) sequenceoperably linked to the DNA encoding the fibronectin based scaffoldprotein.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP Patent Publication No. 73,657. Yeast enhancers also areadvantageously used with yeast promoters.

Transcription from vectors in mammalian host cells can be controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and most preferably Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human .beta.-interferon cDNA in mouse cells under thecontrol of a thymidine kinase promoter from herpes simplex virus.Alternatively, the rous sarcoma virus long terminal repeat can be usedas the promoter.

Transcription of a DNA encoding fibronectin based scaffold proteins byhigher eukaryotes is often increased by inserting an enhancer sequenceinto the vector. Many enhancer sequences are now known from mammaliangenes (globin, elastase, albumin, .alpha.-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thepolypeptide-encoding sequence, but is preferably located at a site 5′from the promoter.

Expression vectors used in eukaryotic host cells (e.g., yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding the polypeptide. Oneuseful transcription termination component is the bovine growth hormonepolyadenylation region. See WO94/11026 and the expression vectordisclosed therein.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the fibronectin based scaffold protein.Examples of protein tags include but are not limited to a histidine tag,a FLAG tag, a myc tag, an HA tag, or a GST tag. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts can be found in Cloning Vectors: A Laboratory Manual,(Elsevier, New York, 1985), the relevant disclosure of which is herebyincorporated by reference.

The expression construct is introduced into the host cell using a methodappropriate to the host cell, as will be apparent to one of skill in theart. A variety of methods for introducing nucleic acids into host cellsare known in the art, including, but not limited to, electroporation;transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (where the vector is aninfectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, orbacterial cells. Suitable bacteria include gram negative or grampositive organisms, for example, E. coli or Bacillus spp. Yeast,preferably from the Saccharomyces species, such as S. cerevisiae, mayalso be used for production of polypeptides. Various mammalian or insectcell culture systems can also be employed to express recombinantproteins. Baculovirus systems for production of heterologous proteins ininsect cells are reviewed by Luckow and Summers, (Bio/Technology, 6:47,1988). Examples of suitable mammalian host cell lines includeendothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3,Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293,293T, and BHK cell lines. Purified fibronectin based scaffold proteinsare prepared by culturing suitable host/vector systems to express therecombinant proteins. For many applications, the small size of thefibronectin based scaffold proteins would make expression in E. coli thepreferred method for expression. The fibronectin based scaffold proteinis then purified from culture media or cell extracts.

Protein Production

Host cells are transformed with the herein-described expression orcloning vectors for protein production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce the fibronectin based scaffold proteinsmay be cultured in a variety of media. Commercially available media suchas Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma)),RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM),(Sigma)) are suitable for culturing the host cells. In addition, any ofthe media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes etal., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866;4,927,762; 4,560,655; or 5,122,469; WO90/03430; WO87/00195; or U.S. Pat.No. Re. 30,985 may be used as culture media for the host cells. Any ofthese media may be supplemented as necessary with hormones and/or othergrowth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

Fibronectin based scaffold proteins disclosed herein can also beproduced using cell-free translation systems. For such purposes thenucleic acids encoding the fibronectin based scaffold protein must bemodified to allow in vitro transcription to produce mRNA and to allowcell-free translation of the mRNA in the particular cell-free systembeing utilized (eukaryotic such as a mammalian or yeast cell-freetranslation system or prokaryotic such as a bacterial cell-freetranslation system).

Fibronectin based scaffold proteins can also be produced by chemicalsynthesis (e.g., by the methods described in Solid Phase PeptideSynthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.).Modifications to the fibronectin based scaffold protein can also beproduced by chemical synthesis.

The fibronectin based scaffold proteins disclosed herein can be purifiedby isolation/purification methods for proteins generally known in thefield of protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, gelfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrent distribution or any combinations ofthese. After purification, fibronectin based scaffold proteins may beexchanged into different buffers and/or concentrated by any of a varietyof methods known to the art, including, but not limited to, filtrationand dialysis.

The purified fibronectin based scaffold protein is preferably at least85% pure, more preferably at least 95% pure, and most preferably atleast 98% pure. Regardless of the exact numerical value of the purity,the fibronectin based scaffold protein is sufficiently pure for use as apharmaceutical product.

Exemplary Uses

In one aspect, the application provides fibronectin based scaffoldproteins labeled with a detectable moiety. The fibronectin basedscaffold proteins may be used for a variety of diagnostic applications.The detectable moiety can be any one which is capable of producing,either directly or indirectly, a detectable signal. For example, thedetectable moiety may be a radioisotope, such as H3, C14, C13, P32, S35,or I131; a fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase.

Any method known in the art for conjugating a protein to the detectablemoiety may be employed, including those methods described by Hunter, etal., Nature 144:945 (1962); David, et al., Biochemistry 13:1014 (1974);Pain, et al., J. Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem.and Cytochem. 30:407 (1982). In vitro methods, include conjugationchemistry well know in the art including chemistry compatible withproteins, such as chemistry for specific amino acids, such as Cys andLys. In order to link a detectable moiety to a fibronectin basedscaffold protein, a linking group or reactive group is used. Suitablelinking groups are well known in the art and include disulfide groups,thioether groups, acid labile groups, photolabile groups, peptidaselabile groups and esterase labile groups. Preferred linking groups aredisulfide groups and thioether groups depending on the application. Forpolypeptides without a Cys amino acid, a Cys can be engineered in alocation to allow for activity of the protein to exist while creating alocation for conjugation.

Fibronectin based scaffold proteins linked with a detectable moiety alsoare useful for in vivo imaging. The polypeptide may be linked to aradio-opaque agent or radioisotope, administered to a subject,preferably into the bloodstream, and the presence and location of thelabeled protein in the subject is assayed. This imaging technique isuseful, for example, in the staging and treatment of malignancies whenthe fibronectin based scaffold protein binds to a target associated withcancer. The fibronectin based scaffold protein may be labeled with anymoiety that is detectable in a subject, whether by nuclear magneticresonance, radiology, or other detection means known in the art.

Fibronectin based scaffold proteins also are useful as affinitypurification agents. In this process, the fibronectin based scaffoldproteins are immobilized on a suitable support, such a Sephadex resin orfilter paper, using methods well known in the art.

Fibronectin based scaffold proteins can be employed in any known assaymethod, such as competitive binding assays, direct and indirect sandwichassays, and immunoprecipitation assays (Zola, Monoclonal Antibodies: AManual of Techniques, pp. 147-158 (CRC Press, Inc., 1987)).

In certain aspects, the disclosure provides methods for detecting atarget molecule in a sample, such as VEGFR2, IGF-IR or EGFR. A methodmay comprise contacting the sample with a fibronectin based scaffoldprotein described herein, wherein said contacting is carried out underconditions that allow fibronectin based scaffold protein-target complexformation; and detecting said complex, thereby detecting said target insaid sample. Detection may be carried out using any technique known inthe art, such as, for example, radiography, immunological assay,fluorescence detection, mass spectroscopy, or surface plasmon resonance.The sample will often be a biological sample, such as a biopsy, andparticularly a biopsy of a tumor, a suspected tumor. The sample may befrom a human or other mammal. The fibronectin based scaffold protein maybe labeled with a labeling moiety, such as a radioactive moiety, afluorescent moiety, a chromogenic moiety, a chemiluminescent moiety, ora hapten moiety. The fibronectin based scaffold protein may beimmobilized on a solid support.

In one aspect, the application provides fibronectin based scaffoldproteins useful in the treatment of disorders. The diseases or disordersthat may be treated will be dictated by the binding specificity of thefibronectin based scaffold protein. The application also providesmethods for administering fibronectin based scaffold proteins to asubject. In some embodiments, the subject is a human. In someembodiments, the fibronectin based scaffold proteins arepharmaceutically acceptable to a mammal, in particular a human. A“pharmaceutically acceptable” polypeptide refers to a polypeptide thatis administered to an animal without significant adverse medicalconsequences. Examples of pharmaceutically acceptable fibronectin basedscaffold proteins include ¹⁰Fn3 domains that lack the integrin-bindingdomain (RGD) and ¹⁰Fn3 domains that are essentially endotoxin or pyrogenfree or have very low endotoxin or pyrogen levels.

In certain embodiments, fibronectin based scaffold proteins, inparticular fibronectin based scaffold proteins that bind to IGF-IR,VEGFR2 and/or EGFR, are useful in treating disorders such as cancer. Incertain embodiments, the fibronectin based scaffold proteins are usefulin treating cancers associated with IGF-IR, VEGFR2 and/or EGFR mutationsor expression levels. In some embodiments, administration of afibronectin based scaffold protein treats an antiproliferative disorderin a subject. In some embodiments, administration of a fibronectin basedscaffold protein inhibits tumor cell growth in vivo. The tumor cell maybe derived from any cell type including, without limitation, epidermal,epithelial, endothelial, leukemia, sarcoma, multiple myeloma, ormesodermal cells. Examples of common tumor cell lines for use inxenograft tumor studies include A549 (non-small cell lung carcinoma)cells, DU-145 (prostate) cells, MCF-7 (breast) cells, Colo 205 (colon)cells, 3T3/IGF-IR (mouse fibroblast) cells, NCI H441 cells, HEP G2(hepatoma) cells, MDA MB 231 (breast) cells, HT-29 (colon) cells,MDA-MB-435s (breast) cells, U266 cells, SH-SY5Y cells, Sk-Mel-2 cells,NCI-H929, RPM18226, and A431 cells. In some embodiments, the fibronectinbased scaffold protein inhibits tumor cell growth relative to the growthof the tumor in an untreated animal. In some embodiments, thefibronectin based scaffold protein inhibits tumor cell growth by 50, 60,70, 80% or more relative to the growth of the tumor in an untreatedanimal. In some embodiments, the inhibition of tumor cell growth ismeasured at least 7 days or at least 14 days after the animals havestarted treatment with the fibronectin based scaffold protein. In someembodiments, another antineoplastic agent is administered to the animalwith the fibronectin based scaffold protein.

In certain aspects, the disclosure provides methods for administeringfibronectin based scaffold protein for the treatment and/or prophylaxisof tumors and/or tumor metastases, where the tumor is selected from thegroup consisting of brain tumor, tumor of the urogenital tract, tumor ofthe lymphatic system, stomach tumor, laryngeal tumor, monocyticleukemia, lung adenocarcinoma, small-cell lung carcinoma, pancreaticcancer, glioblastoma and breast carcinoma, without being restrictedthereto.

In certain aspects, the disclosure provides methods for administeringfibronectin based scaffold proteins for the treatment of cancerousdiseases selected from the group consisting of squamous cell carcinoma,bladder cancer, stomach cancer, liver cancer, kidney cancer, colorectalcancer, breast cancer, head cancer, neck cancer, oesophageal cancer,gynecological cancer, thyroid cancer, lymphoma, chronic leukemia andacute leukemia.

In other embodiments, a fibronectin based scaffold protein binds to atarget involved in inflammatory response and/or autoimmune disorders,such as, for example, tumor necrosis factor (TNF) alpha. Suchfibronectin based scaffold proteins may be useful for treatingautoimmune disorders such as rheumatoid arthritis, ankylosingspondylitis, Crohn's disease, psoriasis and refractory asthma.

Formulation and Administration

The application further provides pharmaceutically acceptablecompositions comprising the fibronectin based scaffold proteinsdescribed herein, wherein the composition is essentially endotoxin orpyrogen free.

Therapeutic formulations comprising fibronectin based scaffold proteinsare prepared for storage by mixing the described proteins having thedesired degree of purity with optional physiologically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of aqueous solutions,lyophilized or other dried formulations. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyidimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrans; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulations herein may also contain more than one active compoundas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The fibronectin based scaffold proteins may also be entrapped inmicrocapsule prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

In certain embodiments, the application provides stable compositions offibronectin based scaffold proteins having a pH of 4.0-6.5. In otherembodiments, the application provides stable compositions of fibronectinbased scaffold proteins having a pH of 4.0-5.5. In other embodiments,the application provides stable compositions of fibronectin basedscaffold proteins having a pH of 5.5. In other embodiments, theapplication provides stable compositions of fibronectin based scaffoldproteins having a pH of 4.0. In particular, the application providesstable compositions of fibronectin based scaffold proteins that havereduced fragmentation and/or low levels of aggregation during storage insolution. As demonstrated in the exemplification section, thefibronectin based scaffold proteins described herein having increasedstability at pH 4.0 while at the same time exhibition decreased levelsof aggregation at pH 4.0 as compared to a pH of 5.5. Such stable,soluble formulations having a pH of 4.0 are particularly suitable forintravenous administration. In some embodiments, the proteinconcentration in such stable formulations is at least 3 mg/mL. Inexemplary embodiments, the protein concentration in such stableformulations is at least 5 mg/mL. In certain embodiments, the proteinconcentration in such stable formulations ranges from 3-10 mg/mL, 3-8mg/mL, 3-6 mg/mL, 3-5 mg/mL, 4-10 mg/mL, 4-8 mg/mL, 4-6 mg/mL, 5-10mg/mL, 5-8 mg/mL, or 5-6 mg/mL. In exemplary embodiments, the stableformulations of fibronectin based scaffold proteins have reducedaggregation relative to an equivalent formulation of a fibronectin basedscaffold protein at a higher pH. For example, the stable formulationsmay exhibit at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, orless aggregation during storage in solution for 4 weeks at pH 4.0relative to the level of aggregation seen during storage of thefibronectin based scaffold protein during storage for 4 weeks at pH 5.5or higher. In certain embodiments, the stable formulations offibronectin based scaffold proteins have less than 10%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1%, or less aggregates after storage at 25° C. for at least4 weeks. In certain embodiments, the stable formulations of fibronectinbased scaffold proteins have less than 7%, 6%, 5%, 4%, 3.5%, 3%, 2% orless fragmentation upon storage in solution for four weeks at pH 4.0 and25° C. In certain embodiments, the stable formulations of fibronectinbased scaffold proteins have less than 5% fragmentation and less than 5%aggregation during storage in solution at 25° C. for at least 4 weeks.In exemplary embodiments, the stable formulations of fibronectin basedscaffold proteins have less than 4% fragmentation and less than 4%aggregation during storage in solution at 25° C. for at least 4 weeks.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the fibronectin based scaffold proteinsdescribed herein, which matrices are in the form of shaped articles,e.g., films, or microcapsule. Examples of sustained-release matricesinclude polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and yethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated proteins remain inthe body for a long time, they may denature or aggregate as a result ofexposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

While the skilled artisan will understand that the dosage of eachfibronectin based scaffold protein will be dependent on the identity ofthe protein, the preferred dosages can range from about 10 mg/squaremeter to about 2000 mg/square meter, more preferably from about 50mg/square meter to about 1000 mg/square meter.

For therapeutic applications, the fibronectin based scaffold proteinsare administered to a subject, in a pharmaceutically acceptable dosageform. They can be administered intravenously as a bolus or by continuousinfusion over a period of time, by intramuscular, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation routes. The protein may also be administered by intratumoral,peritumoral, intralesional, or perilesional routes, to exert local aswell as systemic therapeutic effects. Suitable pharmaceuticallyacceptable carriers, diluents, and excipients are well known and can bedetermined by those of skill in the art as the clinical situationwarrants. Examples of suitable carriers, diluents and/or excipientsinclude: (1) Dulbecco's phosphate buffered saline, pH about 7.4,containing about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9%saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose. The methods of thepresent invention can be practiced in vitro, in vivo, or ex vivo.

Administration of fibronectin based scaffold proteins, and one or moreadditional therapeutic agents, whether co-administered or administeredsequentially, may occur as described above for therapeutic applications.Suitable pharmaceutically acceptable carriers, diluents, and excipientsfor co-administration will be understood by the skilled artisan todepend on the identity of the particular therapeutic agent beingco-administered.

When present in an aqueous dosage form, rather than being lyophilized,the fibronectin based scaffold protein typically will be formulated at aconcentration of about 0.1 mg/ml to 100 mg/ml, although wide variationoutside of these ranges is permitted. For the treatment of disease, theappropriate dosage of fibronectin based scaffold proteins will depend onthe type of disease to be treated, as defined above, the severity andcourse of the disease, whether the fibronectin based scaffold proteinsare administered for preventive or therapeutic purposes, the course ofprevious therapy, the patient's clinical history and response to thefibronectin based scaffold protein, and the discretion of the attendingphysician. The fibronectin based scaffold protein is suitablyadministered to the patient at one time or over a series of treatments.

SEQUENCE LISTING WT Core Sequence (SEQ ID NO: 1)EVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT I core (SEQ ID NO: 2)EVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRT V core (SEQ ID NO: 3)EVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRT Short Tail (SEQ ID NO: 4) EIEKModified Cys Tail (SEQ ID NO: 5) EGSGC Cys Tail (SEQ ID NO: 6) EIEKPCQFn based linker (SEQ ID NO: 7) PSTSTST GS₅ linker (SEQ ID NO: 8)GSGSGSGSGS GS₁₀ linker (SEQ ID NO: 9) GSGSGSGSGSGSGSGSGSGS (GGGGS)₃(SEQ ID NO: 10) GGGGS GGGGS GGGGS (GGGGS)₅ (SEQ ID NO: 11)GGGGS GGGGS GGGGS GGGGS GGGGS G₄SG₄SG₃SG (SEQ ID NO: 12) GGGGSGGGGSGGGSG(SEQ ID NO: 13) GPG (SEQ ID NO: 14) GPGPGPG (SEQ ID NO: 15) GPGPGPGPGPGPA3 linker (SEQ ID NO: 16) PAPAPA PA6 linker (SEQ ID NO: 17)PAPAPAPAPAPA PA9 linker (SEQ ID NO: 18) PAPAPAPAPAPAPAPAPA(SEQ ID NO: 19) MGVSDVPRDL (SEQ ID NO: 20) VSDVPRDL (SEQ ID NO: 21)GVSDVPRDL DK+ VEGFR2/IGF-IR Binder (SEQ ID NO: 22)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRPYQPISINYRTEIDK PSTS TSTVSPVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEID KPCQDK+ EGFR/IGF-IR Binder (SEQ ID NO: 23)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIDK GSGS GSGSGSGSGSGSGSGSVSDVPRDLEVVAATPTSLLISWWAPVDRYQYYRITYGETGGNSPVQEFTVPRDVYTATISGLKPGVDYTITVYAVTDYKPHADGPHTYHESPISINYRTEIDKPCQ DK− EGFR/IGF-IR Binder with GSGC Tail(SEQ ID NO: 24) MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIEK GSGS GSGSGSGSGSGSGSGSVSDVPRDLEVVAATPTSLLISWWAPVDRYQYYRITYGETGGNSPVQEFTVPRDVYTATISGLKPGVDYTITVYAVTDYKPHADG PHTYHESPISINYRTEGSGCDK− EGFR/IGF-IR Binder with EIEKPCQ Tail (SEQ ID NO: 25)MGVSDVPRDLEVVAATPTSLLISVVSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIEK GSG SGSGSGSGSGSGSGSGSVSDVPRDLEVVAATPTSLLISWWAPVPRYQYYRITYGETGGNSPVQEFTVPRDVYTATISGLKPGVDYTITVYAVTDYKPHADGPHTYHESPISINYRTEIEKPCQ (SEQ ID NO: 26) X_(n)SDVPRDL,wherein n = 0, 1 or 2 amino acids, wherein whenn = 1, X is Met or Gly, and when n = 2, X is Met-Gly (SEQ ID NO: 27)X_(n)DVPRDL, wherein n = 0, 1 or 2 amino acids, wherein whenn = 1, X is Met or Gly, and when n = 2, X is Met-Gly (SEQ ID NO: 28)X_(n)VPRDL, wherein n = 0, 1 or 2 amino acids, wherein whenn = 1, X is Met or Gly, and when n = 2, X is Met-Gly (SEQ ID NO: 29)X_(n)PRDL, wherein n = 0, 1 or 2 amino acids, wherein whenn = 1, X is Met or Gly, and when n = 2, X is Met-Gly (SEQ ID NO: 30)X_(n)RDL, wherein n = 0, 1 or 2 amino acids, wherein whenn = 1, X is Met or Gly, and when n = 2, X is Met-Gly (SEQ ID NO: 31)X_(n)DL, wherein n = 0, 1 or 2 amino acids, wherein whenn = 1, X is Met or Gly, and when n = 2, X is Met-Gly (SEQ ID NO: 32)EIEKPSQ (SEQ ID NO: 33) EIEKP (SEQ ID NO: 34) EIEKPS (SEQ ID NO: 35)EIEKPC (SEQ ID NO: 36) EGSGS WT Fibronectin Sequence (SEQ ID NO: 37)VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGPSPASSKPISINYRT ¹⁰Fn3 Core with EIEK Tail(SEQ ID NO: 38) EVVAATPTSLLISW(X)_(x)RYYRITYGETGGNSPVQEFTVP(X)_(y)TATISGLKPGVDYTITVYAVT(X)_(z)PISINYRTEIEK E Core (SEQ ID NO: 39)EVVAATPTSLLISWWAPVDRYQYYRITYGFTGGNSPVQEFTVPRDVYTATISGLKPGVDYTITVYAVTDYKPHADGPHTYHESPISINYRT IGF-IR BC Loop (SEQ ID NO: 40)SARLKVA IGF-IR DE Loop (SEQ ID NO: 41) KNVY IGF-IR FG Loop(SEQ ID NO: 42) RFRDYQ VEGFR2 BC Loop (SEQ ID NO: 43) RHPHFPTVEGFR2 DE Loop (SEQ ID NO: 44) LQPP VEGFR2 FG Loop (SEQ ID NO: 45)DGRNGRLLSI (SEQ ID NO: 46) EIDK (SEQ ID NO: 47) EIDKPCQI-Fn-V(2DK−) with Cys tail (SEQ ID NO: 48)MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIEK PSTS TSTVSDVPRDLEWAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIEK PCQI-Fn-V(2DK−) with ser tail (SEQ ID NO: 49)MGVSPVPRPLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIEK PSTS TSTVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIE KPSQI-GS5-V(2DK−) with ser or cys tail (SEQ ID NO: 50)MGVSDYPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIEK GSGS GSGSGSVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTPGRNGRLLSTPISINYRT EIEKPXQ,wherein X = serine or cysteine I-GS10-V(2DK−) with ser or cys tail(SEQ ID NO: 51) MGVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVPYTITVYAVTRFRPYQPISINYRTEIEK GSGS GSGSGSCSGSGSGSGSVSDVPRPLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLL SIPISINYRTEIEKPXQ,wherein X = serine or cysteine V-Fn-I(2DK−) with ser tail(SEQ ID NO: 52) MGVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIEK PSTSTSTVSPVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIE KPSQV-Fn-I(2DK−) with cys tail (SEQ ID NO: 53)MGVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIEK PSTSTSTVSDVPRDLEVVAATPTSLLISWSARLKVARYYRTTYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIE KPCQV-GS5-I(2DK−) with ser or cys tail (SEQ ID NO: 54)MGVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGIKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIEK GSGSCSGSGSVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRT EIEKPXQ.wherein X = serine or cysteine V-GS10-I(2DK−) with ser or cys tail(SEQ ID NO: 55) MGVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSTPISINYRTEIEK GSGSGSGSGSGSGSGSGSGSVSPVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRD YQPISINYRTEIEKPXQ.wherein X = serine or cysteine VI(DK+) (SEQ ID NO: 56)GVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVPYTITVYAVTRFRDYQPISINYRFEIDK PSTST STVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGLKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIDK PCQ VI(DK-)(SEQ ID NO: 57) GVSDVPRDLEVVAATPTSLLISWSARLKVARYYRITYGETGGNSPVQEFTVPKNVYTATISGLKPGVDYTITVYAVTRFRDYQPISINYRTEIEK PSTST STVSDVPRDLEVVAATPTSLLISWRHPHFPTRYYRITYGETGGNSPVQEFTVPLQPPTATISGEKPGVDYTITVYAVTDGRNGRLLSIPISINYRTEIEK PCQ

EXAMPLES

The invention is now described by reference to the following examples,which are illustrative only, and are not intended to limit the presentinvention. While the invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to one ofskill in the art that various changes and modifications can be madethereto without departing from the spirit and scope thereof.

Example 1. Fibronectin Based Scaffold Proteins

Various fibronectin based scaffold proteins were generated, includingVEGFR2/IGF-IR binders (“V/I binders”) and EGFR/IGF-IR binders (“E/Ibinders”). The following table depicts constructs described herein andtheir corresponding SEQ ID NOs.

TABLE 1 Overview of various V/I and E/I binders. Construct DescriptionSEQ ID NO: Abbreviation DK+ IGF-IR/ A bivalent V/I construct 22 V/I(DK+)VEGFR2 Binder with a C1 tail consisting with EIDKPCQ of SEQ ID NO: 46and a Tail C2 tail consisting of SEQ ID NO: 47 DK+ EGFR/ A bivalent E/Iconstruct 23 E/I(DK+) IGF-IR Binder with a C1 tail consisting withEIDKPCQ of SEQ ID NO: 46 and a Tail C2 tail consisting of SEQ ID NO: 47DK− EGFR/ A bivalent E/I construct 24 E/I(DK−, IGF-IR Binder with a C1tail consisting no C-term) with EGSGC of SEQ ID NO: 4 and a Tail C2 tailconsisting of SEQ ID NO: 5 DK− EGFR/ A bivalent E/I construct 25E/I(2DK−) IGF-IR Binder with a C1 tail consisting with EIEKPCQ of SEQ IDNO: 4 and a Tail C2 tail consisting of SEQ ID NO: 6

SEQ ID NO: 22 is the amino acid sequence of the V/I(DK+) bivalentconstruct that was first described in WO 2009/142773. V/I(DK+) comprisesfibronectin domains that bind to IGF-IR and VEGFR2. The IGF-IR bindingfibronectin core has the sequence set forth in SEQ ID NO: 2 and theVEGFR2 binding fibronectin core has the sequence set forth in SEQ ID NO:3. The two domains are connected by a polypeptide linker derived fromthe amino acid sequence that connects the first and second Fn3 domainsin human fibronectin (SEQ ID NO: 7). The I binding subunit of V/I(DK+)contains a C-terminal extension (C1) having the amino acid sequence SEQID NO: 46, i.e., containing a DK site. The V binding subunit of V/I(DK+)contains a C-terminal extension (C2) having the amino acid sequence ofSEQ ID NO: 47, i.e., containing a DK site.

SEQ ID NO: 23 is the amino acid sequence of the E/I(DK+) bivalentconstruct. E/I(DK+) comprises fibronectin domains that bind to IGF-IRand EGFR. The IGF-IR binding fibronectin core has the sequence set forthin SEQ ID NO: 2 and the EGFR2 binding fibronectin core has the sequenceset forth in SEQ ID NO: 39. The two domains are linked by aglycine-serine polypeptide linker having SEQ ID NO: 9. The I bindingsubunit of E/I(DK+) contains a C-terminal extension (C1) having theamino acid sequence SEQ ID NO: 46, i.e., containing a DK site. The Ebinding subunit of E/I(DK+) contains a C-terminal extension (C2) havingthe amino acid sequence of SEQ ID NO: 47, i.e., containing a DK site.

SEQ ID NO: 24 is the amino acid sequence of the E/I(DK−, no C-term)bivalent construct. The E/I(DK−, no C-term) comprises the IGF-IR core(SEQ ID NO: 2) and EGFR core (SEQ ID NO: 39) linked by a glycine-serinelinker (SEQ ID NO: 9). The IGF-IR binding subunit contains a C-terminalextension (C1) having the amino acid sequence SEQ ID NO: 4, i.e.,containing an EK site rather than a DK site. The EGFR binding subunitcontains a C-terminal extension (C2) having the amino acid sequence ofSEQ ID NO: 5, i.e., lacking a DK site.

SEQ ID NO: 25 is the amino acid sequence of the E/I(2DK−) bivalentconstruct. The E/I(2DK−) comprises the IGF-IR core (SEQ ID NO: 2) andEGFR core (SEQ ID NO: 39) linked by a glycine-serine linker (SEQ ID NO:9). The IGF-IR binding subunit contains a C-terminal extension (C1)having the amino acid sequence SEQ ID NO: 4, i.e., containing an EK siterather than a DK site. The EGFR binding subunit contains a C-terminalextension (C2) having the amino acid sequence of SEQ ID NO: 6, i.e.,containing an EK site rather than a DK site.

Example 2: Expression and Purification of Fibronectin Based ScaffoldProteins Expression of E/I Molecules

E/I bivalent constructs are expressed in E. coli cells in soluble form.The inclusion bodies are recovered by cell disruption andcentrifugation. The E/I proteins are filtered and captured using columnchromatography. The purified protein is then covalently linked to a PEGvia maleimide chemistry at a single cysteine residue. The PEGylatedproduct is then polished using column chromatography and formulatedusing tangential flow filtration.

Expression of V/I Molecules

For expression of V/I bivalent constructs, a nucleotide sequenceencoding the construct is cloned into an inducible expression vector andis expressed into intracellular inclusion bodies in E. coli cells. Cellbank vials generated from a culture of a single plated colony are usedto inoculate a shake flask culture as an inoculum for a large-scalefermentor. Alternatively, a seed fermentor is used for an inoculumculture, depending on the final fermentation volume. The large-scalefermentation contains a growth phase to accumulate biomass and aproduction phase to generate the fibronectin based scaffold proteins.For primary recovery, intracellular inclusion bodies are released fromharvested cells using a microfluidizer and recovered by centrifugation,followed by washes with buffer and water.

The purification process for the bivalent constructs uses aGuanidine-HCl based resolubolization of inclusion bodies, followed byrefolding the protein. The refolded protein is filtered and loaded ontoa cation exchange chromatography column. The product is then purifiedusing a hydrophobic interaction column and the resulting elution pool isPEGylated by the addition of the PEG reagent to produce PEGylatedprotein.

The PEGylated protein is then purified over a second cation exchangechromatography column. The elution is concentrated to a target proteinconcentration and then exchanged into the formulation buffer usingultrafiltration/diafiltration (UF/DF). The UF/DF product is filteredusing a final 0.22 μm filter. The filtered product is then filled intovials to produce the final drug product.

Example 3: Effects of Protein Concentration on V/I Protein Stability

The effects of protein concentration on physical (aggregation) andchemical (fragmentation) stability of purified V/I(DK+) (SEQ ID NO: 22)were examined. V/I(DK+) protein was formulated in 10 mM succinic acid,5% sorbitol at pH 5.5. V/I protein concentration was either at 3 mg/mLor at 5 mg/ml. Samples were stored at 4° C. for a period of 12 months,with samples being collected and analyzed at 1 month, 6 weeks, 2 months,3 months, 6 months, 9 months and 12 months.

The amount of aggregation is measured by assessing the percentage oftotal protein that has formed aggregates (measured as High MolecularWeight (“HMW”) species) over time. Aggregation was determined using SizeExclusion-High Performance Liquid Chromatography (SE-HPLC) analysis toassess the levels of HMW over time. SE-HPLC analysis was conducted usinga Superdex 200 10/300 GL column, with a mobile phase of 0.2M potassiumphosphate, 0.15 M sodium chloride, 0.02% sodium azide, pH 6.8. Flow ratewas 0.5 mL/min with detection at 280 nm. The effects of proteinconcentration on aggregation of V/l protein over time (0-12 months) isdepicted in FIG. 1. V/I(DK+) aggregates at a rate of 0.3%/month at aconcentration of 3 mg/mL. Higher protein concentration (5 mg/ml) leadsto a faster aggregation rate.

Fragmentation is measured by assessing percentage of total protein thathas been fragmented, or “clipped”, over time. Levels of clipped proteinwere determined by utilizing Reversed Phase-High Performance LiquidChromatography (RP-HPLC). RP-HPLC was performed using a Varian PLRP-Scolumn (4.6*250 mm, 300 Å pore size, 5 μm particle size). Separation ofthe various species is achieved via a gradient comprised ofwater/acetonitrile/trifluoroacetic acid. Flow rate was 1.0 mL/min. Dualdetection was conducted at 280 nm (for protein-related species) and withevaporative light scattering (ELS, for PEG-related species). FIG. 2demonstrates that fragmentation of V/I(DK+) was found to be lessdependent on protein concentration than was aggregation. Thefragmentation rate for V/I(DK+) was ˜0.1%/month at 4° C.

Based on these aggregation and fragmentation data, a formulation ofV/I(DK+) having a protein concentration of 3 mg/mL would be preferredover a concentration of 5 mg/mL in order to minimize aggregation and toensure sufficient stability for one year.

Example 4: Effects of pH on V/l Protein Stability

The effects of pH on physical and chemical stability of purifiedV/I(DK+) (SEQ ID NO: 22) were examined. V/I(DK+) protein was formulatedin 50 mM sodium chloride, with the buffer component being 20 mM sodiumacetate (for pH 4 and 5) or 20 mM sodium phosphate (for pH 6 and 7), andwas stored at 25° C.

Samples were collected once per week for a period of four weeks andevaluation of aggregation was carried out using SE-HPLC analysis asdescribed in Example 3. The effects of pH on aggregation of V/I(DK+)protein over time (0-4 wks) are depicted in FIG. 3. The lowestaggregation rate was observed in the samples having the lowest pH tested(pH 4.0). The highest aggregation rate was observed in the sampleshaving the highest pH tested (pH 7.0).

Samples were collected once per week for a period of four weeks andevaluation of fragmentation was carried out using SE-HPLC analysis asdescribed in Example 3. The effects of pH on fragmentation of V/I(DK+)protein is depicted in FIG. 4. While a low pH (pH 4.0) was found toprevent aggregation over time of the V/I(DK+) protein (FIG. 3), low pHwas found to lead to an increase in protein fragmentation of theV/I(DK+) protein (FIG. 4).

To identify clip sites in the fragmented V/I(DK+) protein, liquidchromatography-Mass Spectrometry (LC-MS) was performed. V/I(DK+) proteinwas formulated in 10 mM sodium acetate, 150 mM sodium chloride, pH 5.5,at 5 mg/mL protein concentration. LC-MS was performed according to theRP-HPLC method described in Example 3 followed by coupling to a ThermoLTQ ion trap mass spectrometer (MS). The HPLC eluent was split 1:5 with0.2 mL/min flow diverted into the MS. On-line detection at 280 nm wasmaintained. FIG. 5 depicts the LC-MS data from this experiment anddemonstrates that several aspartate (D) residues are involved infragmentation, with D95 and D200 being the predominant sites at whichfragmentation occurs. It should be noted that while there are several Dresidues throughout the V/I(DK+) protein (e.g. D5, D9, D110), only theD95 and D200 residues are immediately followed by a lysine residue. “VIdes Met” indicates a cleavage event occurring at the maleimide bond ofthe PEG conjugation as a result of heat stress.

Based on these experiments, aggregation and fragmentation are bestbalanced by fixing the formulation pH at 5.5, however, neitherdegradation pathway (aggregation and fragmentation) could be eliminatedby relying on this pH level alone.

Example 5: Evaluation of Aggregation and Fragmentation on E/I Proteins

To confirm that the aggregation and fragmentation instability issuesobserved in V/I(DK+) (SEQ ID NO: 22) were issues common to otherstructurally related bivalent constructs, the aggregation andfragmentation properties of E/I(DK+) (SEQ ID NO: 23) were also assessedat several pH levels.

E/I(DK+) was formulated in 10 mM succinic acid, 5% sorbitol and at pH4.0, 4.5 or 5.5. E/I(DK+) was formulated into the desired formulationvia tangential flow filtration (TFF) using a 30 kD MWCO membrane. Atleast six dia-volumes of buffer were exchanged to achieve the finalformulation. Concentration of the resulting protein was verified by A280and adjusted to 5 mg/mL with additional formulation buffer. FormulatedE/I(DK+) was sterile filtered in a laminar flow hood, and filled intosterilized glass vials for stability monitoring. The vials were cappedand crimped, followed by placement into temperature-controlledincubators at 4, 25 and 37° C.

Aggregation rate of E/I(DK+) was assessed by performing SE-HPLCanalysis. SE-HPLC was performed using a Shodex KW404-4F HPLC column(4.6*250 mm, 300 Å pore size, 5 μm particle size) and a mobile phasecomprised of 10 mM succinic acid/3% sorbitol/0.4M arginine at pH 5.5.Flow rate was 0.35 mL/min and detection was conducted at 280 nm. FIG. 6shows that, similar to V/I(DK+), the aggregation rate of E/I(DK+) washigher for samples stored at higher pH levels at 25° C. than for samplesstored at lower pH levels at 25° C. The same data trends were alsoobserved for samples stressed at 37° C.

The effect of protein concentration on aggregation rate of E/I(DK+) wasalso assessed. Similar to V/I(DK+), Table 2 illustrates that higherconcentrations (7 mg/ml) of E/I(DK+) were associated with a higherpercentage of aggregate formation (lower percentage of monomers) after 4weeks. Table 2 also demonstrates that by reducing pH along with proteinconcentration, the percent of aggregation can be further decreased.

TABLE 2 Protein Conc. Formulation pH % Monomer 7.5 mg/mL 5.5 94.30% 5.0mg/mL 5.5 97.70% 5.0 mg/mL 4.0 99.20%

Fragmentation of the E/I(DK+) was assessed by performing RP-HPLCanalysis as described in Example 3. FIG. 7 shows that, similar toV/I(DK+), the fragmentation rate of E/I(DK+) was highest for samplesstored at lower pH levels at 25° C. then for samples stored at higher pHlevels at 25° C. The same data trends were also observed for samplesstressed at 37° C.

To identify clip sites in the fragmented E/I(DK+) protein, LC-MS wasperformed as described in Example 4. E/I(DK+) was formulated in 10 mMsuccinic acid, 5% sorbitol, pH 4.0, at 5 mg/mL protein concentration andwas maintained at 25° C. to induce protein stress. FIG. 8 depicts theLC-MS data from this experiment and demonstrates that several aspartate(D) residues are involved in fragmentation, including D95 and D218 (thehomologous position to D200 in V/I(DK+)). Fragmentation was alsoobserved at D199. The D199 site is specific to these E/I molecules andis not found in V/I molecules, as it is located within the FG bindingloop of the EGFR-binding region.

Example 6: Evaluation of Aggregation and Fragmentation in DK Minus E/IVariants

Various E/I binders (SEQ ID NOs: 23-25) were formulated and theirphysical (aggregation) and chemical (fragmentation) stability wascompared to each other under identical conditions (see Table 1). Basedupon the characterization of the D95 and D218 clipped sites observed inE/I(DK+) (Example 5), and based on the fact that these DK clip sites arelocated in the structurally nonessential C-terminal tails, two differentE/I constructs were generated in which the C-terminal tail DK sites wereremoved or substituted.

The E/I(DK-t) molecule (SEQ ID NO: 23) is the control E/I binder thatcontains a C-terminal tail comprising DK sites (D95 and D218) after eachof the two binding domains. The physical and chemical stability of thismolecule was characterized in Example 5. The E/I(DK−, no C-term)molecule (SEQ ID NO: 24) does not contain any DK sites. In thismolecule, the aspartate at position 95 was mutated to a glutamic acid,and the EIDKPCQ tail (SEQ ID NO: 47) was replaced with an EGSGC tail(SEQ ID NO: 5). The E/I(2DK−) molecule (SEQ ID NO: 25) also does notcontain any DK sites. In this molecule the aspartates at positions 95and 218 have been replaced with glutamic acids. V/I(DK+) was included inthis study as a control.

The E/I proteins (SEQ ID NOs: 23-25) were formulated in 10 mM succinicacid, 5% sorbitol at pH 4.0. In addition, E/I(DK+) (SEQ ID NO: 23) andV/I(DK+) (SEQ ID NO: 22) were formulated in 10 mM succinic acid, 5%sorbitol at pH 5.5. Surfactant was not found to be necessary based on apreliminary surfactant screen. E/I(DK+) was formulated into the desiredformulation via tangential flow filtration (TFF) using a 30 kD MWCOmembrane. At least six dia-volumes of buffer were exchanged to achievethe final formulation. Concentration of the resulting protein wasverified by A280 and adjusted to 5 mg/mL with additional formulationbuffer. Each formulated bivalent construct was sterile filtered in alaminar flow hood, and filled into sterilized glass vials for stabilitymonitoring. The vials were capped and crimped, followed by placementinto temperature-controlled incubators at 4, 25 and 37° C.

Aggregation rate for the different E/I molecules was determined byperforming SE-HPLC as described according to Example 5 and the resultsfrom this experiment are illustrated in FIG. 9. As expected, the rate ofaggregation is significantly higher at pH 5.5 than at pH 4.0 forE/I(DK+) at 25° C. Based on the slopes seen in FIG. 9, the rate ofaggregation for E/I(DK+) is approximately 7-fold higher at pH 5.5 thanat pH 4.0 at 25° C. For the two E/I molecules lacking DK sites, theaggregation rate was unaffected at pH 4.0. E/I(DK+) and V/I(DK+)displayed similar aggregation rates at pH 5.5.

Fragmentation rate for the different E/I molecules was determined byperforming RP-HPLC according to Example 3 and the results from thisexperiment are illustrated in FIG. 10. In terms of clipping. FIG. 10shows lower clip rates at pH 5.5 than at pH 4.0 for E/I(DK+) at 25° C.Among the molecules represented in FIG. 10, the highest clip rate wasseen in E/I(DK+) at pH 4.0, which was significantly minimized when theformulation pH was increased to pH 5.5. However, as described above,aggregation rate was highest at pH 5.5 for this molecule. For the othertwo E/I molecules lacking DK sites, the clip rates at pH 4.0 decreasedby approximately 3-fold as compared to E/I(DK+) at the same pH. V/I(DK+)displayed a higher degree of fragmentation as compared to E/I(DK+) atthe same pH (pH 5.5) indicating that although the V/I(DK+) and E/I(DK+)molecules are similar, the V/I(DK+) molecule is more susceptible tofragmentation.

Characterization of clipped sites for E/I(DK−, no C-term), as comparedto the clipped sites for E/I(DK+), was performed using LC-MS asdescribed in Example 4. The two different E/I Binders were eachformulated in 10 mM succinic acid, 5% sorbitol, pH 4.0, at 5 mg/mLprotein concentration and were maintained at 25° C. to induce proteinstress. FIG. 11 depicts the LC-MS data from this experiment anddemonstrates that the fragmentation profiles differ between the twodifferent E/I binders. The predominant aspartate (D) residues involvedin fragmentation in E/I(DK+) were D95, D218 and D199. By contrast, thepredominant aspartate residues involved in fragmentation in E/I(DK−, noC-term) were D199, D82 and D193. However, as illustrated in Table 3below, the percentage of total clips was nearly halved in E/I(DK−, noC-term) after 4 weeks as compared to E/I(DK+) after this same period(3.8% compared to 7.5%). These results indicate that E/I(DK−, no C-term)is associated with reduced fragmentation as compared to E/I(DK+).

Characterization of clipped sites in E/I(2DK−) was performed using LC-MSas described in Example 4. E/I(2DK−) was formulated in 10 mM succinicacid, 5% sorbitol, pH 4.0, at 5 mg/mL protein concentration and wasmaintained at 25° C. to induce protein stress. FIG. 12 depicts the LC-MSdata from this experiment and demonstrates that similar to E/I (DK−, noC-term), the predominant aspartate residues involved in fragmentation inE/I(2DK−) were D199, D82 and D193. Also, as illustrated in Table 3below, the percentage of total clips was more than halved in E/I(2DK−)after 4 weeks, as compared to E/I (DK+) after this same period (3.5%compared to 7.5%). These results indicate that E/I(2DK−) is associatedwith reduced fragmentation as compared to E/I(DK+).

TABLE 3 Amount and location of fragmentation for various E/I bindersafter four weeks of storage at 25° C. SEQ ID Clip Sites Identified %Total Clips at 4 wks Construct NO (in order of intensity) (value at T =0) E/I(DK+) 23 D218K 7.5% D199G (0.5%) D95K E/I(DK−, 24 D199G 3.8% noC-term) D193Y (1.4%) D82Y E/I(2DK−) 25 D199G 3.5% D193Y (0.9%) D82Y

As discussed above, the D199 site is located within the FG binding loopof the EGFR-binding region. As such, D199 likely is necessary forbinding function and will be difficult to remove from the E/I(2DK−) orE/I(DK−, no C-term) molecules.

The exact mechanism of clipping at the aspartic acid sites observed inV/I(DK+) and E/I(DK+) is not clear. Based on apparent pKa values ofthree aspartic acids in glucagon as measured by NMR methods, Joshi etal. (Journal of Pharmaceutical Sciences, 94 (9), 2005) proposed severalmechanisms for the cleavage reaction at aspartic acid sites. Withoutwishing to be bound by theory, it is possible that some of the proposedmechanisms involve cyclization of the aspartic acid side chain to form afive-member ring, followed by nucleophilic attack on the peptidecarbonyl which then leads to peptide bond cleavage. By substitutingaspartic acid with glutamic acid, it is possible that the ring formationdoes not occur as readily due to steric hindrance, thus preventingpeptide bond cleavage at that location.

Example 7: Evaluation of Aggregation and Fragmentation in DK Minus V/IVariants

The stability of two VEGFR-IGFR (VI) fibronectin based scaffold proteinshas been compared. The first construct is VI(DK+) (SEQ ID NO: 56), andthe second construct VI(DK−) (SEQ ID NO: 57) contains substitution ofaspartic acid with glutamic acid at positions 94 and 199 of SEQ ID NO:56.

Both molecules were formulated at 3 mg/mL protein concentration, in 10mM succinic acid, 5% sorbitol, at pH 4.0, 4.5 and 5.5. Limited stabilityof these formulations was performed at 4 and 25° C. for up to twomonths, with periodic time points pulled for analysis by SE-HPLC andRP-HPLC. In addition, LC-MS characterization was performed on the2-month 25° C. samples in order to determine the exact clipped sites inboth proteins.

Effects of pH on Aggregation Rate in VI Fibronectin Based ScaffoldProteins

Based on experience with past fibronectin based scaffold proteins, lowpH formulations have been recognized to provide the best biophysicalstability for these molecules (i.e., lower aggregation). In the currentstudy, the two VI constructs demonstrate the same trend as that seenbefore, as illustrated in FIG. 13. Although the starting levels ofaggregates are slightly different between the two molecules, the ratesobserved over the stability period are very similar at each given pH,with the aggregation rates for both molecules showing the same order, pH5.5>>pH 4.5>pH 4.0.

Effects of pH on Clip Rate in VI Fibronectin Based Scaffold Proteins

Based on experience with past fibronectin based scaffold proteins, lowpH formulations have been recognized to provide the least chemicalstability for these proteins, if sites susceptible to clipping exist inthe protein sequence. In past stability studies conducted for VI(DK+)(SEQ ID NO: 56), numerous clip sites have been identified, with clippingat D94 and D199 being the most severe. In the current study, the two VIconstructs demonstrate the same trend as seen before, as illustrated inFIG. 14, with the pH effects on clip rate being worse for VI(DK+) thanfor VI(DK−). While the clip rate follows the general trend of pH 4.0>pH4.5>pH 5.5 for both molecules, VI(DK−) exhibits much less difference inits clip rate across all three pH values.

From the stability data trends shown in FIGS. 13 and 14, the clip rateper week for VI(DK+) increases by 3.3-fold when the formulation pH isdecreased from 5.5 to 4.0, whereas for the VI(DK−) molecule, this rateincreases by 1.6-fold, due to the elimination of the major clip sites attwo positions. Therefore, with these amino acid substitutions, the cliprate in the VI fibronectin based scaffold protein has decreased by about50% over the same stability period when the pH 4 formulation is used. Onthe other hand, the aggregation rates per week for VI(DK+) and VI(DK−)decrease by 86- and 216-fold, respectively, when the formulation pH isdecreased from 5.5 to 4.0. The pH effect on aggregation rate, therefore,is more drastic than on the clip rate in VI fibronectin based scaffoldproteins.

Identification of Clipped Sites by LC-MS

Structural characterization of the observed clipped sites in VI(DK+) andVT(DK−) has been performed by LC-MS. The overlaid RP-HPLC chromatogramof the clip region from the 25° C./2-month time point can be seen inFIG. 15 and peak identification is summarized in Table 4.

TABLE 4 Summary of peak identification by mass spectrometry. Residueshighlighted in red indicate the aspartic acids that exist in theoriginal VI sequence which have been replaced by glutamic acids in theVI(DK−) construct. Peak Area Peak Area Structure % Among Structure %Among of Clips All Clips of Clips All Clips Peak # in VI(DK+) in VI(DK+)in VI(DK−) in VI(DK−) 1 G1-D105 11.6 G1-D105 37.8 2 G1-D81 3.1 G1-D817.9 3 G1-D94 24.7 G1-R80 36.3 4 G1-D199 43.0 G1-D179 14.1 5 G1-K200 3.7T15-D81 3.9 6 (G1-D199)-18 5.1 n/a n/a 7 (G1-D199)-35 3.2 n/a n/a 8D109-Q203 1.9 n/a n/a

While some clips remain identical between the two molecules, the majorclip sites at D94 and D199 have been eliminated in VI(DK−), bringing thelevel of total clips to 6.9% after 2 months of stress at 25° C. On theother hand, the total clips in VI(DK+) remained high at 16.0% over thesame stability period. Other clips at low level have also beenidentified in VI(DK−), which are not found in VI(DK+).

TABLE 5 Comparison of Clip Rates in VI(DK+) and VI(DK−). Rate forVI(DK+) Rate for VI(DK−) Formulation pH (% per week) (% per week) 4.01.6650 0.6350 4.5 1.0375 0.4330 5.5 0.4975 0.3925

The clip rate of VI(DK+) at pH 4.0 was 1.6 fold and 3.3 fold faster thanthe clip rate of VI(DK+) at pH 4.5 and 5.5, respectively. The clip rateof VI(DK−) at pH 4.0 was 1.5 fold and 1.6 fold faster than the clip rateof VI(DK−) at pH 4.5 and 5.5, respectively.

TABLE 6 Comparison of Aggregation Rates in VI(DK+) and VI(DK−). Rate forVI(DK+) Rate for VI(DK−) Formulation pH (% per week) (% per week) 4.00.0225 −0.0075 4.5 0.4450 0.4050 1.9400 1.9400 1.6200

The agreggation rates of VI(DK+) at pH 4.5 and 5.0 were 4.4 fold and 86fold faster, respectively, than the agreggation rate of VI(DK+) at pH4.0. The agreggation rates of VI(DK−) at pH 4.5 and 5.0 were 4.0 foldand 216 fold faster, respectively, than the agreggation rate of VI(DK−)at pH 4.0.

The stability results obtained on two versions of the VI fibronectinbased scaffold proteins demonstrate the effectiveness and benefits ofselective substitutions of problematic aspartic acids to glutamic acids,for the purpose of eliminating specific chemical degradation in thefibronectin based scaffold protein. With the major chemical degradationeliminated through this approach, better biophysical stability infibronectin based scaffold proteins may now be achieved throughformulation at the lower pH. The results from the current study with VI,as well as those previously obtained from EI bi-functional fibronectinbased scaffold proteins, demonstrate the necessity in eliminatingcertain aspartic acid residues known to be prone to clipping, which inturn allows for formulations at acidic pH to be used in order tomaximize the biophysical stability in the fibronectin based scaffoldproteins.

Materials and Methods

Formulation: Each molecule was formulated into the desired formulationsvia tangential flow filtration (TFF) using a 30 kD MWCO membrane. Atleast six dia-volumes of buffer were exchanged to achieve the finalformulation. Concentration of the resulting protein was verified by A280and adjusted to 3 mg/mL with additional formulation buffer.

Vial Fill and Stability: Each formulated protein was sterile filtered ina laminar flow hood, and filled into sterilized glass vials forstability monitoring. The vials were capped and crimped, followed byplacement into temperature-controlled incubators at 4 and 25° C.

Analytical Methods:

Size Exclusion HPLC (SE-HPLC): The analysis was conducted using a ShodexKW404-4F HPLC column (4.6*250 mm, 300 Å pore size, 5 μm particle size)and a mobile phase comprised of 10 mM succinic acid/3% sorbitol/0.4Marginine at pH 5.5. Flow rate was 0.35 mL/min and detection wasconducted at 280 nm.

Reversed Phase HPLC (RP-HPLC): The analysis was performed using a VarianPLRP-S column (4.6*250 mm, 300 Å pore size, 5 μm particle size).Separation of the various species is achieved via a gradient comprisedof water/acetonitrile/trifluoroacetic acid. Flow rate was 1.0 mL/min.Dual detection was conducted at 280 nm (for protein-related species) andwith evaporative light scattering (ELS, for PEG-related species).

LC-MS: Characterization of clipped sites was performed using a JupiterC18 column (4.6*250 mm, 300 Å pore size, 5 μm particle size) coupled toa Thermo LTQ ion trap mass spectrometer (MS). The HPLC eluent was split1:5 with 0.2 mL/min flow diverted into the MS. On-line detection at 280nm was maintained.

INCORPORATION BY REFERENCE

All documents and references, including patent documents and websites,described herein are individually incorporated by reference to into thisdocument to the same extent as if there were written in this document infull or in part.

1-46. (canceled)
 47. A method of treating a hyperproliferative diseasecomprising administering to a subject in need thereof a therapeuticallyeffective amount of a fibronectin-based protein dimer comprising a firstfibronectin type III tenth (¹⁰Fn3) domain and a second ¹⁰Fn3 domain,wherein each of the first ¹⁰Fn3 domain and the second ¹⁰Fn3 domain: (i)comprises an AB loop, a BC loop, a CD loop, a DE loop, an EF loop, and aFG loop, wherein the first and second ¹⁰Fn3 domains have at least oneloop selected from the BC, DE, and FG loops with an altered amino acidsequence relative to the sequence of the corresponding loop of the human¹⁰Fn3 domain having the amino acid sequence of SEQ ID NO: 1; (ii)comprises an amino acid sequence having at least 60% identity to SEQ IDNO: 1 and binds to insulin-like growth factor 1 receptor (IGF-IR),vascular endothelial growth factor receptor 2 (VEGFR2), or epidermalgrowth factor receptor (EGFR); and (iii) comprises a C-terminal tailconsisting of an amino acid sequence selected from the group consistingof SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO:
 35. 48. The method ofclaim 47, wherein one or both of the first ¹⁰Fn3 domain and the second¹⁰Fn3 domain further comprised) an N-terminal extension comprising asequence selected from the group consisting of: M, MG, G, and any of SEQID NOs: 19-21 and 26-31.
 49. The method of claim 47, wherein the first¹⁰Fn3 domain and the second ¹⁰Fn3 domain bind to different targets. 50.The method of claim 47, wherein the first ¹⁰Fn3 domain and the second¹⁰Fn3 domain are connected by a polypeptide linker comprising 1-30 aminoacids.
 51. The method of claim 50, wherein the linker is selected fromthe group consisting of: a glycine-serine based linker, aglycine-proline based linker, a proline-alanine linker, and an Fn-basedlinker.
 52. The method of claim 47, wherein the protein dimer has lessthan 4% fragmentation during storage in solution at pH 4.0 for at least4 weeks.
 53. The method of claim 47, further comprising one or morepharmacokinetic (PK) moieties selected from: a polyoxyalkylene moiety, ahuman serum albumin binding protein, sialic acid, human serum albumin,transferrin, and an Fc fragment.
 54. The method of claim 47, wherein theprotein dimer is administered by an intravenous, intramuscular,subcutaneous, or oral route.
 55. The method of claim 47, furthercomprising administering one or more additional therapeutic agents. 56.The method of claim 47, wherein the hyperproliferative disorder is acancer selected from the group consisting of: squamous cell carcinoma,bladder cancer, stomach cancer, liver cancer, kidney cancer, colorectalcancer, breast cancer, head cancer, neck cancer, esophageal cancer,gynecological cancer, thyroid cancer, lymphoma, chronic leukemia, andacute leukemia.
 57. A method of inhibiting tumor cell growth in asubject comprising administering to the subject a therapeuticallyeffective amount of a fibronectin-based protein dimer comprising a firstfibronectin type III tenth (¹⁰Fn3) domain and a second ¹⁰Fn3 domain,wherein each of the first ¹⁰Fn3 domain and the second ¹⁰Fn3 domain: (i)comprises an AB loop, a BC loop, a CD loop, a DE loop, an EF loop, and aFG loop, wherein the first and second ¹⁰Fn3 domains have at least oneloop selected from the BC, DE, and FG loops with an altered amino acidsequence relative to the sequence of the corresponding loop of the human¹⁰Fn3 domain having the amino acid sequence of SEQ ID NO: 1; (ii)comprises an amino acid sequence having at least 60% identity to SEQ IDNO: 1 and binds to insulin-like growth factor 1 receptor (IGF-1R),vascular endothelial growth factor receptor 2 (VEGFR2), or epidermalgrowth factor receptor (EGFR); and (iii) comprises a C-terminal tailconsisting of an amino acid sequence selected from the group consistingof SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO:
 35. 58. The method ofclaim 57, wherein one or both of the first ¹⁰Fn3 domain and the second¹⁰Fn3 domain further comprised) an N-terminal extension comprising asequence selected from the group consisting of: M, MG, G, and any of SEQID NOs: 19-21 and 26-31.
 59. The method of claim 57, wherein the first¹⁰Fn3 domain and the second ¹⁰Fn3 domain bind to different targets. 60.The method of claim 57, wherein the first ¹⁰Fn3 domain and the second¹⁰Fn3 domain are connected by a polypeptide linker comprising 1-30 aminoacids.
 61. The method of claim 60, wherein the linker is selected fromthe group consisting of: a glycine-serine based linker, aglycine-proline based linker, a proline-alanine linker, and an Fn-basedlinker.
 62. The method of claim 57, wherein the protein dimer has lessthan 4% fragmentation during storage in solution at pH 4.0 for at least4 weeks.
 63. The method of claim 57, further comprising one or morepharmacokinetic (PK) moieties selected from: a polyoxyalkylene moiety, ahuman serum albumin binding protein, sialic acid, human serum albumin,transferrin, and an Fc fragment.
 64. The method of claim 57, furthercomprising administering one or more additional therapeutic agents. 65.The method of claim 57, wherein the tumor is selected from the groupconsisting of: brain tumor, tumor of the urogenital tract, tumor of thelymphatic system, stomach tumor, laryngeal tumor, monocytic leukemia,lung tumor, small-cell lung carcinoma, pancreatic tumor, glioblastoma,and breast tumor.
 66. A method of detection comprising (i) contacting asample with a fibronectin-based protein dimer under conditions thatallow the fibronectin-based protein dimer to form a complex with atarget, and (ii) detecting the complex, wherein the fibronectin-basedprotein dimer comprises a first fibronectin type III tenth (¹⁰Fn3)domain and a second ¹⁰Fn3 domain, wherein each of the first ¹⁰Fn3 domainand the second ¹⁰Fn3 domain: (i) comprises an AB loop, a BC loop, a CDloop, a DE loop, an EF loop, and a FG loop, wherein the first and second¹⁰Fn3 domains have at least one loop selected from the BC, DE, and FGloops with an altered amino acid sequence relative to the sequence ofthe corresponding loop of the human ¹⁰Fn3 domain having the amino acidsequence of SEQ ID NO: 1; (ii) comprises an amino acid sequence havingat least 60% identity to SEQ ID NO: 1 and binds to a target molecule;(iii) comprises a C-terminal tail consisting of an amino acid sequenceselected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, andSEQ ID NO: 35.